Immunogenic peptides and their use in immune disorders

ABSTRACT

The present invention provides novel peptides and homologues thereof. The peptides of the invention comprise (i) a T-cell epitope of an antigen (self or non-self) with a potential to trigger an immune reaction presented by a class II major histocompatibility complex (MHC) determinant and recognised by CD4+ T cell more specifically of an allergen or auto-antigen, coupled, optionally through the use of a linker to (ii) an amino acid sequence having a reducing activity, such as a thioreductase sequence. The peptides of the invention have been shown to be useful a medicine, more in particular for the prevention or treatment of immune disorders, more specifically of allergic disorders or autoimmune diseases. The present invention thus provides for the use of said peptides for the manufacture of a medicament for the prevention or treatment of an immune disorder and further provides for methods of treatment or preventing immune disorders by using said peptides. The present invention also provides for compositions comprising said peptides.

FIELD OF THE INVENTION

The present invention relates to immunogenic peptides and their use intherapies for suppressing allergies and autoimmune disorders.

BACKGROUND OF THE INVENTION

The mammalian immune system is a complex network that serves to protecta subject from external and internal endangering factors. However, insome circumstances, this complex protection mechanism sustains or itselfbecomes a cause of disorders, mostly with chronic implications, withinthe subject. Many such immune disorders exist, two important ones beingthe allergic diseases and the autoimmune disorders. Allergic diseases,conventionally described as type-1 mediated diseases or IgE-mediateddiseases, have seen their prevalence almost doubled over the last 20years. Clinical manifestations of allergic diseases include bronchialasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andanaphylactic reactions to insect bites or drugs. The economical burdenrelated to the care of allergic patients is steadily increasing over theyears. As an example, the cost linked to prescription of allergytreatment in the US is anticipated to reach around 10 billion US dollarsin 2006. There is currently no curative therapy for such diseases, whichare kept under control by allergen eviction whenever possible, and/or bysymptomatic therapy using bronchodilators, anti-histamines,corticosteroids and immunomodulators such as cyclosporine. Allergendesensitisation, which consists in regular administration of allergensto which the patient is sensitised, has shown efficacy in allergicrhinitis, but remains controversial in asthma and atopic dermatitis.Some clinical symptoms, such as those related to food allergens, cannotbe treated by desensitisation.

Autoimmunity is the failure of an organism to recognise its ownconstituent parts (down to the sub-molecular level) as “self”, whichresults in an immune response against its own cells and tissues. Anydisease that results from such an aberrant immune response is termed anautoimmune disease. Prominent examples are Systemic Lupus Erythematosus(SLE), Sjögren's syndrome and Rheumatoid Arthritis (RA). Autoimmunediseases are broadly classified into two categories, namely systemicdiseases and organ-specific diseases. The precise aetiology of systemicautoimmune diseases is not identified. In contrast, organ-specificautoimmune diseases are related to a specific immune response includingB and T cells, which targets the organ and thereby induces and maintainsa chronic state of local inflammation. Examples of organ-specificautoimmune diseases include type 1 diabetes, myasthenia gravis,thyroiditis, multiple sclerosis, celiac disease, inflammatory boweldiseases, atherosclerosis, adrenalitis, polyendocrine syndromes,gastritis, pernicious anemia, ocular diseases such as uveitis, and innerear diseases such as cochleitis.

Autoimmune reactions are thus directed to own cells or tissues, moreparticularly to “auto-antigens” i.e. antigens (of proteins) that arenaturally present in the mammalian organism. In this mechanism,auto-antigens are recognised by B- and/or T-cells which activate theimmune system to attack the tissue comprising the auto-antigen. It iswell recognised that suppression of the immune system is beneficial andin some cases leads to partial or complete recovery of organ function insome instances. This kind of therapy is however not effective for allorgan-specific autoimmune disease and up to date immune suppression cannot be achieved in an antigen-specific manner. Current therapy makes useof non-specific immune suppression obtained by the use ofcorticosteroids and immunosuppressive agents, all exhibiting significantside effects related to the lack of specificity, thereby limiting theiruse and their overall efficacy.

Interestingly, for reasons that are far from being understood, theincidence of autoimmune diseases has doubled over the last 20 years,much in parallel to the increase observed in allergic diseases. Again,the cost related to the treatment of autoimmune diseases has increasedenormously in recent years, adding a further argument to the need for anew form of therapy.

In the prior art, T-cell epitopes of allergens have been used fordesensitisation purposes. Allergen-derived peptides containing one or afew T cell epitope(s) are used in animal experiments and in human beingsin an attempt to inhibit specific T cell activation and induce a stateof T cell unresponsiveness, such as described in the patent applicationWO93/08279. One human application of this concept is the administrationof a peptide derived from the sequence of T cell epitopes present on theFel d I allergen, by subcutaneous injections in cat-sensitiveindividuals (Wallner & Gefter (1994) Allergy 49, 302-308). Analternative, complementary approach of this concept has also been usedin animal experiments. The peptides used were modified in such a manneras to keep the ability to bind to MHC-class II determinants on specificB cells, but these peptides lost their capacity to activate thecorresponding T cells (O'Hehir et al. (1991) Int. Immunol. 3, 819-826).

The screening of the allergen Der p 2 of house mite with a set ofoverlapping peptides from this protein shows that one specific peptidep21-35 comprises a T-cell epitope which behaves as universal epitope andcould be a suitable candidate for T cell anergy induction (Wu et al.(2003) J. Immunol. 169, 1430-2435, WO0170263). In a related publicationit was shown that this peptide and derivatives thereof have an epitopespecific effect on CD4+ CD25+ mediated apoptosis of antigen presenting Bcells (Janssens et al. (2003) J. Immunol. 171, 4604-4612). Theidentification of this peptide however required an exhaustive screeningof the allergen and there is no indication that for each and everyantigenic protein, such a peptide with an apoptosis-inducing effect canbe identified.

It is clear that there is a need for novel strategies or drugs for theprevention or treatment of immune diseases like allergic or autoimmunediseases, which are more effective, more specific, haveless-side-effects, are curative instead of merely treating symptoms ofdisease and are easily accessible, more particularly at low cost. Moreparticularly, for allergic diseases, there is a need for the developmentof new forms of therapy that are specific for the concerned allergens,that are safe and produce long-lasting beneficial effects.

SUMMARY OF THE INVENTION

The present invention relates to novel immunogenic peptides withcytotoxic activity. The peptides of the invention comprise (i) at leastone T-cell epitope of an antigen (self or non-self) with a potential totrigger an immune reaction, coupled, optionally through the use of alinker to (ii) an organic compound having an reducing activity, such asa thioreductase sequence and furthermore optionally comprise (iii) anendosome targeting amino acid sequence.

In one aspect the present invention provides isolated immunogenicpeptides derived from an antigenic protein comprising an artificialsequence comprising a T cell epitope of the antigenic protein and motifC—X(2)-[CST] or [CST]-X(2)-C, which motif has reducing activity,resulting in a specific response by T cells when contacted with thispeptide.

In particular embodiments isolated immunogenic peptides derived from anantigenic protein are provided comprising an artificial sequencecomprising a T cell epitope of the antigenic protein and motif C—X(2)-C,whereby the motif is positioned either adjacent to the epitope, orseparated from the epitope within the artificial sequence by a linker ofat most 7 amino acids. In further particular embodiments, the isolatedimmunogenic peptide derived from an antigenic protein comprise anartificial sequence comprising a T cell epitope of the antigenic proteinand motif C—X(2)-[CST] or [CST]-X(2)-C, whereby the motif is positionedeither adjacent to the epitope, or separated from the epitope by alinker of at most 7 amino acids within the artificial sequence andwhereby the motif does not naturally occur within a region of 11 aminoacids N-terminally or C-terminally of the T-cell epitope in the proteinfrom which the epitope is derived. Further particular embodimentsprovide isolated immunogenic peptides derived from an antigenic proteincomprising an artificial sequence comprising a T cell epitope of theantigenic protein and motif C—X(2)-[ST] or [ST]-X(2)-C, whereby motif ispositioned either adjacent to the epitope, or separated from the epitopeby a linker of at most 7 amino acids within the artificial sequence, andwhereby in those peptides where the motif is C—X(2)-S or S—X(2)-C, the Tcell epitope does not comprise the sequence EPCIIHRGKP [SEQ ID. NO: 1]of the p21-35 peptide of Der p 2. Further particular embodimentscorrespond to immunogenic peptides as described above, wherein for thosepeptides wherein the motif is C—X(2)-S or S—X(2)-C the antigenic proteinis not Der p 2.

Further particular embodiments of the invention relate to immunogenicpeptides such as those described hereinabove, which further comprise,linked to the artificial sequence a late endosomal targeting sequence.

Further particular embodiments of the invention relate to immunogenicpeptides such as those described hereinabove, comprising the motifpositioned N-terminally of the epitope.

Further particular embodiments of the invention relate to immunogenicpeptides such as those described hereinabove, wherein the artificialsequence has a length of between 12 and 19 amino acids.

In particular embodiments of the invention, immunogenic peptides areprovided such as those described hereinabove, wherein X in the motif isnot Tyr, or another bulky amino acid such as Trp or Phe. Additionally oralternatively in particular embodiments, at least one of X in the motifis Gly, Ala, Ser or Thr. Additionally or alternatively in particularembodiments, at least one X in the motif is H or P.

In particular embodiments of the invention, immunogenic peptides areprovided such as those described hereinabove, wherein Cysteine in thecorresponding motif is methylated. In the case of the motif C—X(2)-C oneor both Cysteines in the motif can be methylated.

The immunogenic peptides of the present invention are envisaged to be ofuse for the generation of an immuno-suppressive effect, whereby thetargeted immunosuppressive effect will determine the nature of theantigenic protein from which the epitope is derived. In particularembodiments of the immunogenic peptides described hereinabove, theantigenic protein is an auto-antigen, more particularly an auto-antigenselected from the group consisting of thyroglobulin, thyroid peroxidase,TSH receptor, insulin (proinsulin), glutamic acid decarboxylase (GAD),tyrosine phosphatase IA-2, myelin oligodentrodycte protein, heat-shockprotein HSP65.

In further particular embodiments of the immunogenic peptides describedhereinabove, the antigenic protein is an allergen, more particularly anallergen selected from the group consisting of Betula Bet v1 allergen,Bovine beta-lactoglobulin and Der p1.

A further aspect of the present invention relates to the therapeutic andprophylactic use of the immunogenic peptides described hereinabove.Accordingly, the present invention provides peptides such as thosedescribed above, for use as a medicament, and pharmaceuticalcompositions comprising one or more of the peptides described above,optionally comprising a pharmaceutically acceptable carrier.

A further aspect of the present invention relates to the use of thepeptides described hereinabove in the treatment and prevention ofauto-immune disorders. As indicated above, the peptides are described tohave both therapeutic and prophylactic effect thereby allowing areduction in the occurrence of, a reduction of the occurrence and/orseverity of relapses and/or the prevention of the auto-immune disease.Thus the present invention provides the immunogenic peptides describedhereinabove, for use in the treatment and prevention of an autoimmunedisorder. Specific embodiments of the auto-immune disorders envisaged inthe context of the present invention include but are not limited tomultiple sclerosis, spontaneous insulin-dependent diabetes andautoimmune thyroiditis.

Yet a further aspect of the present invention relates to the use of thepeptides described hereinabove in the treatment and prevention ofallergic conditions. As indicated above, the peptides are described tohave both therapeutic and prophylactic effect thereby allowing areduction in the occurrence and/or severity of the allergic conditionand/or the prevention of the allergic condition and/or a reduction inthe symptoms of the allergic condition. More particularly the presentinvention provides peptides as described herein for use in the treatmentand prevention of an allergic condition selected from the groupconsisting of dust mite allergy, milk allergy and birch pollen allergy.

Yet a further aspect of the present invention provides methods forpreparing a peptide of an antigenic protein capable of elicitingcytolytic CD4+ T cell activity said method comprising the steps of (a)providing a peptide sequence consisting of a T-cell epitope of theantigenic protein, and linking to this peptide sequence a sequencecomprising motif C—X(2)-[CST] or [CST]-X(2)-C, such that the motif andthe epitope are either adjacent to each other or separated by a linkerof at most 7 amino acids. In particular embodiments, the motif is motifC—X(2)-C. In particular embodiments of methods according to this aspectof the invention, the T-cell epitope is an epitope of an antigenicprotein which does not naturally comprise the motif C—X(2)-[CST] or[CST]-X(2)-C within a region of 11 amino acids N-terminally orC-terminally of the T-cell epitope and the T-cell epitope is linked tomotif C—X(2)-[CST] or [CST]-X(2)-C. In yet other particular embodimentsof methods according to this aspect of the invention, where the T-cellepitope comprises the sequence EPCIIHRGKP [SEQ ID. NO: 1] of the p21-35peptide of Der p 2, the motif is not C—X(2)-S or S—X(2)-C. In furtherparticular embodiments, the antigenic protein is not Der p 2.

In particular embodiments methods of the present invention furthercomprise modifying the sequence of the peptide thus obtained bymodifying the amino acids in the epitope, thereby ensuring that in themodified peptide the sequence of the epitope is modified such thatability to fit into the MHCII cleft is maintained. Such modificationsinclude amino acid substitutions but also include changes in amino acidchain such as modifications encountered in post-translationalmodifications of amino acids or even non-naturally occurring non-naturalamino acid side chains.

Yet a further aspect of the invention relates to methods for preparingan isolated immunogenic peptide of an antigenic protein capable ofeliciting cytolytic CD4+ T cell activity, which methods comprise thesteps of identifying within the antigenic protein, a sequence comprisinga T cell epitope flanked, in the antigenic protein, by motifC—X(2)-[CST] or [CST]-X(2)-C within a region of 11 amino acidsN-terminally or C-terminally of said T-cell epitope, and generating apeptide comprising this sequence as an isolated peptide of between 12and 19 amino acids. It has not previously been demonstrated that in thisway, peptides with particular immunogenic properties can be generated.In particular embodiments, the antigenic protein is not Der p 2.

According to further particular embodiments methods according to thisaspect of the invention further comprise modifying the sequence of saidpeptide by modifying the amino acids in the motif and/or by modifyingthe number of amino acids between the motif and the epitope and/or bymodifying the epitope sequence, thereby ensuring that in said modifiedpeptide:

-   -   the ability of the T cell epitope to fit into the MHCII cleft is        maintained,    -   the motif is conserved and    -   said motif and said epitope remain adjacent to each other or        separated by a linker of at most 7 amino acids.

According to further particular embodiments methods according to thisaspect of the invention further comprise the step of attaching a lateendosomal targeting sequence to the peptide obtained as described above.

Yet a further aspect of the invention relates to methods of identifyinga population of cytotoxic Tregs. In particular embodiments, methods areprovided which comprise determining that the cells express CD4, do notexpress IL-10 or TGF-beta, and express Krox-20 and produce granzymes (inparticular Granzymes B and C) and Fas ligand.

In further particular embodiments, methods according to this aspect ofthe invention comprise determining one or more of the followingcharacteristics, when compared to non-cytotoxic Tregs:

-   a) an increased expression of surface markers including CD103,    CTLA-4, FasL and ICOS upon activation,-   b) a high expression of CD25, expression of CD4, ICOS, CTLA-4, GITR    and low or no expression of CD127 (IL7-R),-   c) the expression of transcription factor T-bet and/or egr-2    (Krox-20) but not of the transcription repressor Foxp3,-   d) a high production of IFN-gamma and no or only trace amounts of    IL-10, IL-4, IL-5, IL-13 or TGF-beta.-   e) an increased expression of markers including FasL and granzymes B    and C upon activation.

In further particular embodiments, methods according to this aspect ofthe invention comprise determining that these cells do not respond tothe activation by TCR recognition.

Yet a further aspect of the invention provides methods for obtaining apopulation of antigen-specific regulatory T cells with cytotoxicproperties. In a particular embodiment, methods according to this aspectcomprise the steps of:

-   -   providing peripheral blood cells,    -   contacting said cells with an immunogenic peptide as described        before and    -   expanding said cells in the presence of IL-2.

In a further particular embodiment these methods comprise administeringan immunogenic peptide according to the present invention to a subject,and isolating from said subject, the antigen-specific regulatory T cellswith cytotoxic properties.

Yet a further aspect of the present invention relates to populations ofregulatory T cells with cytotoxic properties, obtainable (and/oridentifiable) by the methods of the present invention described above.

Yet a further aspect of the present invention provides for the use ofthe population of T regulatory cells described hereinabove in thetreatment and prevention of an allergic condition or an auto-immunedisorder.

In particular embodiments of this aspect of the invention, methods areprovide which comprise the steps of:

-   -   providing peripheral blood cells of the subject to be treated,    -   contacting the cells with an immunogenic peptide as described        herein,    -   expanding the cells, and    -   administering the expanded cells to the subject to be treated.

More particularly in methods provided herein an immunogenic peptide isused of which the T-cell epitope is derived from an antigenic proteininvolved in the disease process to be treated. Most particularly theantigen is a dominant antigen.

The invention further provides methods of treating or preventing anauto-immune disorder in a subject, comprising the steps of administeringone or more immunogenic peptides as described herein to said subject.Moreover the invention provides methods for treating or reducing thesymptoms an allergic condition in an subject, comprising the steps ofadministering one or more of the immunogenic peptides as describedherein to said subject. More particularly, in methods provided herein,immunogenic peptides are used of which the T-cell epitope is derivedfrom an antigenic protein involved in the disease process to be treated.Most particularly the antigen is a dominant antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the capacity of p21-35 of Der p 2 to reduce disulfidebridges in the insulin reduction assay (turbidimetric assay) (dashedline: control; solid line with triangles; B4 peptide; solid line withsquares: Trx (thioredoxin)

FIG. 2 shows the increase of uptake of p21-35 by antigen-presentingcells (assayed by apoptosis of target Wehi B cells) by addition of asubdominant T cell epitope (grey line with squares: T-B peptide (p21-35linked to the minor T epitope (p830-844) of tetanus toxin; black linewith triangles: p21-35 peptide).

FIG. 3 shows the effect of mutating positions 21 to 24 in P21-35 intoAlanine on Treg clone proliferation (3H thymidine incorporation (panelB) and Wehi cell lysis (panel C) according to an embodiment of theinvention. The motif and the residues forming the MHC-class II bindingcleft in p21-35 are indicated in panel A. (neg: without peptide; a21:Cys21Ala mutation; a22: His22Ala mutation; a23: Gly23Ala mutation, a24:Ser24Ala mutation).

FIG. 4 shows the effect of preimmunisation with T-B in an in vivo mousemodel for allergy upon injection with Der p 2 protein according to anembodiment of the invention. The “Der p 2 model” is a control group ofmice, Tbalum is an experimental group pretreated with T-B peptide.

Panel A: Amount (expressed as total numbers) of macrophages, eosinophilsand lymphocytes in control and experimental group.Panel B: Amount of eosinophils, lymphocytes and goblet cells expressedusing an intensity scoring system from 0 to 6.Panel C: Airway hyperreactivity in control and experimental group.Hyperreactivity is measured by calculating the area under the curve(AUC) for PenH values obtained by exposing mice to increasingconcentrations of methacholine.

FIG. 5 shows the effect of p21-35 on the oxidative metabolism of cognateTreg cells measured by cell sorting of Carboxy-H2DCFDA labelled cells.Panel A: PBS (negative control); Panel B: p21-35 peptide; Panel C;tert-butyl hydroperoxide (positive control).

FIG. 6 shows the cytotoxic properties of a Treg line (G121) on the WEHIB cell line used as an antigen-presenting cell upon addition of thep21-35 peptide, indicated as percentage cell lysis (diamonds: WEHI+Tcells, squares: WEHI cells+T cells+p21-35 peptide, triangles Tcells+WEHI cells pre-loaded with the p21-35 peptide).

FIG. 7 shows the suppression by Treg cell clones on the activation of Tcells specific for another epitope on the same antigen, or specific foranother antigen by cytotoxicity according to an embodiment of theinvention.

Panels A and B: T cell line specific for Der p1.Panels C and D: T cell line specific for peptide p71-85 of Der p 2.Panels A and C shows the proliferation of the cell lines prior toincubation with a cytotoxic Treg clone specific for peptide 21-35.Panels B and D shows the proliferation of the cell lines afterincubation with a cytotoxic Treg clone specific for peptide 21-35.

FIG. 8 shows that Treg cells of the invention have a characteristicphenotypic profile.

The Figure shows cytokine production of 4 peptide p21-35 specific Tregclones derived from mice treated with the peptide p21-35, peptide T-B asin FIG. 4 (left panel). Supernatants of cell culture were analysed forcytokine content after four days of stimulation with antigen-presentingcells (irradiated splenocytes from naïve mice, 10⁵ cells) and peptidep21-35 (2 μg/ml, 200 μl). It can be seen that Treg clones mainlyproduced IFN-G and only trace amounts of TNF-a and IL-10. The rightpanel shows that m-RNA analysis of such Treg cells, transcripts fortranscription repressor Foxp3 were not detected, but transcripts forT-bet, Granzyme A and Granzyme B were strongly expressed.

FIG. 9 shows the expression of various cellular markers of four p21-35specific T cell clones at rest using fluororescence-activated cellsorting (Facs).

FIG. 10 shows a schematic overview of the generation of T cell cloneswith cytotoxic properties after stimulation with a control peptide(targeting signal—Der p1 epitope) and an experimental peptide (targetingsignal —CFGS—Der p 1epitope) according to an embodiment of theinvention. “n” is the total number of T cell clones, “Lytic” is thenumber of these clones which have the capacity to lyse WEHI cells.

FIG. 11 shows the elicitation of antigen-specific regulatory T cellsusing epitope sequences with mutated residues according to an embodimentof the invention.

Panel A: Induction of apoptosis (indicated as Annexin-V expression onCD19+ gated cells using Wehi cells preloaded with p21-35 (triangle),p21-35met (p21-35 peptide with methylated cysteine) (cross) or mp 21-35(fusion peptide of a minor epitope of tetanus toxoid and P21-35)(square), and cocultured for 24 hours with the G121 cytolytic Tregclone. Results representative of at least 3 experiments;Panel B: Suppression of spleen CD4+ T cell proliferation (measured as^([3])H thymidine incorporation) induced by entire Der p 2 protein(black), p21-35met (p21-35 peptide with methylated cysteine) (white) ora mixture of p830 (peptide p830-844 of Tetanus toxoid) and p21-35met(grey). Histograms are for average cpm±s.e.m. from 6 mice testedindividually in triplicates;Panel C: Production of cytokines by spleen CD4+ T cells pre-treated withthe peptides indicated in panel b. All three cell populations werestimulated with intact Der p2. Histograms are for averageconcentration±s.e.m. from 6 mice tested individually;Panel D: Processing and presentation of p21-35met, mp 21-35 and Der p 2assessed using adherent splenocytes as APC and an effector p21-35specific CD4+ T cell clone (G221N). APC were pre-treated with theindicated inhibitors. Proliferation of T cells is shown as a stimulationindex. Bars represent s.e.m. values of triplicate cultures.

FIG. 12 shows the phenotypic characterisation of cytolytic Treg clonesobtained with mp 21-35Asn (fusion peptide of tetanus toxoid peptide andmutated p21-35 (Ile28Asn) according to an embodiment of the invention.The Treg clones in alum were tested for surface marker expression andintracellular CTLA-4.

Panel A: Expression of surface markers (Facs) from a Treg clone;Panel B: intracellular detection of Foxp3, T-bet, granzyme B (Grz-B),perforin and surface CD127 using fluorescence-labelled specificantibodies (black). Control staining with an isotype-matched antibody isalso shown (white).Panel C: RT-PCR of Grz-A and Grz-B mRNA transcripts detected 12 daysafter the last stimulation in 4 cytolytic clones. Lanes 1, 3, 4 and 5show Treg clones and lane 2 a control p21-35 specific CD4+ effector Tcell clone. Beta-actin was used as a control.Panel D: ELISA detection of cytokines in 4 clones after 3 days ofstimulation with irradiated T cell-depleted splenocytes obtained fromnaïve mice and loaded with mp 21-35Asn

FIG. 13 shows the induction of apoptosis in antigen-presenting cellsaccording to an embodiment of the invention.

Part A: Left panel: Incubation (18 hours) of splenic B cells preloadedwith p21-35 with R3TB7 T cell clone (ratio 2/1).Right panel: Incubation (18 hours) of splenic B cells preloaded withp21-35 with a control CD4+ effector T cell clone. White areas withdashed lines represent caspase-3 expression in B cells cultured withoutT cells; grey areas with solid lines show caspase-3 expression in thepresence of the cytolytic Treg clone (left panel) or the CD4+ effectorclone (right panel). Staining with Ab against cleaved Caspase 3. Datarepresent evaluation from a minimum of 3 independent experimentsPart B: Left panel: Dendritic cells (CD11c+ dendritic cells activated byLPS) Right panel: WEHI cellsBoth cell types are loaded with p21-35 and co-cultured with R3TB7 Treg(black areas) or a control non-cytolytic clone of the same specificity(G221N) (white areas). Apoptosis is measured using an Ab against cleavedCaspase 3. Cell count refers to the number of surviving WEHI cells after18 hours of incubation. Data representative of two experiments. The %suppression indicated is the % of suppression of Wehi growth by R3TB7.Part C: Left panel: Incubation of WEHI cells (loaded with p21-35met andincubated with R3TB7 Treg (1/1 ratio)) with anti-FasL antibody.Right panel: Incubation of WEHI cells (loaded with p21-35met andincubated with R3TB7 Treg (1/1 ratio)) with a peptide antagonistic ofGZ-B (Z-AAD-CMK) (black squares) or a chemical inhibitor of serineproteases (DCIC: 3,4-dichloroiso-coumarin) (white squares).Part D: Left panel: Apoptosis measured by Annexin V expression (greyarea) of dendritic cells (CD11c+ cells loaded with p21-35 and incubatedin the presence of G121 Treg at a 1/1 ratio for 18 hours)Right panel: as in the left panel, but DC and Treg cells are separatedby a semi-permeable membrane in a transwell culture system.(White area (hidden in panel D) represents annexin V binding on DCwithout cytotoxic T cells). Results are representative of twoindependent experiments.Part E: Two populations of WEHI cells were labelled with either 80 nM or300 nM of CFSE and incubated for 1 hour with p21-35 or p71-85,respectively. The cells were then incubated with G121 Treg (left panel)or with control CD4+ effector T cells (right panel). Binding of annexinV was analysed by flow cytometry after an incubation of 18 hours.

FIG. 14: Suppression of bystander T cells by cytotoxic T cells accordingto an embodiment of the invention.

Part A: FACS analysis of CFSE stained cells of CD4+CD25(−) spleen cells,incubated with T cell-depleted splenocytes used as APC, 1 μg/ml anti-CD3antibody, 1 μg/ml p21-35, and a cytolytic Treg cell line. A 1/3 ratio ofTregs over CD4+CD25(−) T cells was used in uncoated polystyrene cultureplates with V-shaped wells to optimise cell contact.left panels: cytolytic Treg cell line G121middle panels: cytolytic Treg cell line R3TB7right panels: control cell culture wherein cytolytic Treg are replacedby unlabeled CD4+CD25(−) splenocytes (right panels). The number ofcells, cell divisions and cell size were evaluated by Facs after livecells gating.top panels: incubation for 48 hoursbottom panels: incubation for 72 hoursPart B: Analysis for Annexin V binding on CFSE labelled CD4+CD25(−) Tcells after coculture with R3TB7 Treg cell line after 18 hours (middlepanel) and after 24 hours (right panel). The left panel shows a controlculture (24 hours) without cytolytic Tregs.Part C: Experiment with the experimental settings of the lower panels ofpart A, but CFSE divisions were evaluated after 72 h of co-culture(except for the left panel), without EGTA and without cytolytic Tregcell line (left panel), in the presence of 2 mM EGTA (middle panel) or 4mM (right panel) of EGTA.Part D: Labelling of a Der p 1-specific Th2 cell clone with CFSE andcultivation for 72 h with T cell-depleted splenocytes loaded withcognate peptide (amino acids 114 to 128 from Der p 1). Gating was madeon propidium iodide negative CFSE positive cells. The left panel showsproliferation determined on a leftward fluorescence shift in thepresence of the same unlabeled Th2 cells (ratio 1/1). The second panelfrom left shows results obtained upon addition of G121 Treg (1/1 ratiowith the Th2 clone) plus p21-35 (1 μg/ml) to the culture, in thepresence of a control antibody. The next 3 panels (left to right) showthe effects obtained when antibodies to FasL, GITR or Lag3 were addedfrom the start of coculture with the cytolytic clone. Each antibody wasused at 10 μg/ml. Percentages are for the proportion of PI negative CFSElabelled within total population of CFSE cells.Part E: Incubation of a CFSE-labelled Th1 clone specific to p71-85 ofDer p 2 for 72 hours with an equal number of the same unlabeled clone(left panel). Proliferation is shown as a fluorescence shift to theleft. This experiment was repeated but with replacing the unlabeledclone by a cytolytic Treg in a single well (middle panel), or in twowells separated by a transwell membrane (right panel). Cell ratio was1/1. Results are representative of three independent experiments.Histograms represent PI negative CFSE labelled cells.Part F: Incubation of T cell-depleted splenocytes with a CFSE-labelledp71-85-specific Th1 clone for 18 hours. The p71-85 peptide was added toactivate the clone in each case except for the control (far left panel).To this culture, a p830-844 control cell clone was added with itsspecific peptide (left panel), or a cytolytic clone (R3TB7) with orwithout the p21-35 peptide (far right and right panels, respectively).Cell ratio was 1/1. Density dot plots are shown. Results are expressedas average FSC values (upper value, black) and percentages of blastingCFSE cells (lower value, grey). Blast formation was calculated from cellsize on CFSE-positive cells in the living lymphocyte gate (establishedfrom FSC/SSC plots). The resting lymphocyte gate was adjusted to theregion showing the highest density of non-stimulated cells (far leftpanel). Data are representative of at least 3 experiments.

FIG. 15 shows the localisation of cytolytic Tregs to the lungs inallergen-exposed mice and the in vivo induction of apoptosis by antigenpresenting B cells according to an embodiment of the invention.

A: Splenic B cells isolated by magnetic beads from naïve BALB/c micewere transduced with a retrovirus vector encoding p21-35 and the gp75protein. The left panel represents B cells incubated for 18 hours with acytolytic T cell clone (ratio 2/1). The right panel represents the sameassay but carried out with a control CD4+ effector cell. Dashed curvesrepresent anti-caspase-3 staining in B cells cultured without T cells.B: 5×10⁶ p21-35 transduced B cells were administered IV to each mouse(n=6), followed 5 days later by 5×10⁵ cytolytic Tregs. Two weeks later,mice were sacrificed and cells were prepared from the spleen and lungsby density gradient purification and CD19+ selection using magneticbeads. The presence of mRNA coding for the retroviral construct used togenerate transgenic B cells was detected by PCR. A group of mice (n=6)received the transduced B cells but no cytolytic T cells, was treatedlikewise. Six mice in each group were analysed and representativeresults are shown in lanes A for spleens of 2 mice treated with thecytolytic Tregs and in lanes B for 2 mice of the control group.C: 5×10⁵ Vβ8.1+ cytolytic Tregs, or a V8.1+ control T cell clone, wereadministered IV to naïve BALB/c mice, followed 24 h later by three nasalinstillations with 100 microgram Der p 2. Two weeks later, mice weresacrificed and lung lymphocytes prepared by density gradientcentrifugation. The proportion of cells expressing Vbeta.8.1 wascalculated by Facs within the population of CD4+ cells. Results areexpressed as average %±s.e.m. from 6 mice in each group. % CD4 is thepercentage of CD4 within the total lymphocytic population; % Vb8.1 isthe percentage of cells expressing Vb8.1 within the total CD4 cellpopulation.

FIG. 16 shows the prevention (a-f) and suppression of (g-l) experimentalasthma by cytolytic Treg clones according to an embodiment of theinvention. Panels A to F show data from mice treated with mp21-35Asn-specific cytolytic Tregs (clones T1 and T3) before IPsensitisation with Der p 2. (The “control T cell clone” is specific forpeptide 830-844 of tetanus toxoid, “Der P 2 model” refers to experimentswherein no cells are administered to the cells.)

In panels G to L mice where treated with the above cell lines after IPsensitisation with Der p 2Panels A and G show total BALF cell numbers;Panels B and H shows differential BALF cell counts;Panels C and I shows BALF cytokines as measured by ELISA;Panels D and J shows a semi-quantitative scoring for lung infiltrationby eosinophils and lymphocytes.Panels E and K show goblet cell counting. Results are expressed as theproportion (%) of goblet cells within the population of epithelial cellsafter PAS staining;Panels F and L show airway hyper-reactivity evaluated by inhalation ofincreasing concentrations of methacholine. PenH values were determinedusing a whole body plethysmograph. Results are shown as “Area Under TheCurve” (AUC) for PenH values. For comparison, AUC values obtained innaïve mice in an independent experiment are shown (naïve). This groupgives background values in both prevention and suppression assays.Data represent results from a minimum of 5 mice per group. Barsrepresent mean±s.e.m. *P≦0.05, **P≦0.01 compared (one-tailed P value) tothe Der p 2 model.

FIG. 17 shows the induction of apoptosis by effector CD4 T cell linespecific for Der p1 T cell epitope (114-128), labelled with CFSE andincubated with APC loaded with corresponding peptide (114-128) (upperpanel) according to an embodiment of the invention. The lower panelshows baseline mortality (40%) when an identical number of effectorcells (unlabelled) replaced the regulatory T cell clone.

FIG. 18A shows BALF differential cell count was after nasalinstillations with either 100 μg Der p1 (model) or NaCl (negative). Micewere adoptively transferred with cytolytic T cells either prior to(prevention) or after (suppression) the first series of nasalinstillation with Der p 1. Bars represent mean±SEM, *p<0.05; **p<0.01 ascompared to negative group.

FIG. 18B shows the histology scoring after adoptive transfer ofcytolytic T cells according to an embodiment of the invention. Scoreswere established for eosinophil, lymphocyte and plasmocyte infiltratesusing a scale from 0 to 6 (no lesion to massive infiltrate). Micereceived 2 series of 3 nasal instillations with either 100 μg Der p 1(model) or NaCl (negative). Mice were adoptively transferred withcytolytic T cells either prior to (prevention) or after (suppression)the first series of nasal instillation with Der p 1. Bars representmean±SEM, *p<0.05; **p<0.01; ***p<0.001 as compared to model group.

FIG. 19A shows the production of TNF-alpha (black histograms) andIFN-gamma (grey histograms) of cytolytic clone stimulated with APC usingno peptide, wildtype p21-35 peptide (C-x-x-S), or Ser24Cys mutatedp21-35 (C-x-x-C) according to an embodiment of the invention.

FIG. 19B shows semi-quantitative PCR detection of Granzyme (lanes 1 to3) and Fas-L (lanes 6 to 8) transcripts. A cytotoxic clone wasstimulated with APC loaded with wildtype p21-35 (lanes 1, 6), Ser24Cysmodified p21-35 (lanes 2, 7) or without peptide (lanes 3, 8). Lanes 4and 5 are molecular weight markers.

FIG. 20 shows apoptosis of effector T cells labelled with CFSE andco-cultured with APC cells according to an embodiment of the invention.The bars indicate unlabelled T effector cells and wild type MOG peptide:T_(effect) wtMOG; Tr cells and unmodified MOG peptide: Tr wtMOG; Trcells and modified MOG peptide containing the thioredoxin sequence: TrCSMOG). CD4+ CD25+ T cells were obtained from animals immunised with MOGpeptide containing a thioredoxin consensus sequence (CSMOG). EffectorCD4+CD25− cells were obtained from EAE animals (T_(effect)).

FIG. 21 shows effect on the injection modified MOG peptide on thedevelopment of MS in a mouse model. (0: no disease, 1: limp tail, 2:limp tail and loss of weight higher than 10%, 3: partial paralysis ofhind limbs, 4: complete paralysis of hind limbs) Model: 3 C57BL/6 micereceived, at day 0, SC injection of 100 μg MOG peptide/400 μgMycobacterium butyricum in CFA and ip injection of 300 ng Bortetellapertussis in NaCl. At day +2, a second injection of B. pertussis wasgiven. Adoptive transfert: 3 mice received iv injection with 500,000Treg, 24 h before disease induction as in model group.

FIG. 22 shows the clinical scoring of mice in with and withoutprevention by injection of MOG peptide in a model for MultipleSclerosis.

FIG. 23 shows the in vitro induction of apoptosis in CFSE labelledpolyclonal CD4 cells from two NOD mice splenocytes. These cells wereco-cultured with APC loaded with GAD65 peptide 524-543 [SEQ ID. NO: 34]together with polyclonal CD4 cells purified from modifiedpeptide-treated NOD mice. The figure shows staining of target CD4 cellswith 7-AAD and Annexin V-PE. The table represent the percentage ofdouble positive cells (dead cells).

DETAILED DESCRIPTION Definitions

The term “peptide” as used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can in a particular embodiment comprisenon-amino acid structures (like for example a linking organic compound).Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino-acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification.

The term “antigen” as used herein refers to a structure of amacromolecule, typically protein (with or without polysaccharides) ormade of proteic composition comprising one or more hapten (s) andcomprising T cell epitopes. The term “antigenic protein” as used hereinrefers to a protein comprising one or more T cell epitopes. Anauto-antigen or auto-antigenic protein as used herein refers to a humanor animal protein present in the body, which elicits an immune responsewithin the same human or animal body.

The term “food or pharmaceutical antigenic protein” refers to anantigenic protein naturally present in a food or pharmaceutical product,such as in a vaccine.

The term “epitope” refers to one or several portions (which may define aconformational epitope) of an antigenic protein which is/arespecifically recognised and bound by an antibody or a portion thereof(Fab′, Fab2′, etc.) or a receptor presented at the cell surface of a Bor T cell lymphocyte, and which is able, by said binding, to induce animmune response.

The term “T cell epitope” in the context of the present invention refersto a dominant, sub-dominant or minor T cell epitope, i.e. a part of anantigenic protein that is specifically recognised and bound by areceptor at the cell surface of a T lymphocyte. Whether an epitope isdominant, sub-dominant or minor depends on the immune reaction elicitedagainst the epitope. Dominance depends on the frequency at which suchepitopes are recognised by T cells and able to activate them, among allthe possible T cell epitopes of a protein.

In particular embodiments, a T cell epitope is an epitope recognised byMHC class II molecules, which consists of a sequence of +/−9 amino acidswhich fit in the groove of the MHC II molecule. Within a peptidesequence representing a T cell epitope, the amino acids in the epitopeare numbered P1 to P9, amino acids N-terminal of the epitope arenumbered P−1, P−2 and so on, amino acids C terminal of the epitope arenumbered P+1, P+2 and so on, as illustrated in FIG. 3A.

The term “homologue” as used herein with reference to the epitopes usedin the context of the invention, refer to molecules having at least 50%,at least 70%, at least 80%, at least 90%, at least 95% or at least 98%amino acid sequence identity with the naturally occurring epitope,thereby maintaining the ability of the epitope to bind an antibody orcell surface receptor of a B and/or T cell. Particular embodiments ofhomologues of an epitope correspond to the natural epitope modified inat most three, more particularly in at most 2, most particularly in oneamino acid.

The term “derivative” as used herein with reference to the peptides ofthe invention refers to molecules which contain at least the peptideactive portion (i.e. capable of eliciting cytolytic CD4+ T cellactivity) and, in addition thereto comprises a complementary portionwhich can have different purposes such as stabilising the peptides oraltering the pharmacokinetic or pharmacodynamic properties of thepeptide.

The term “sequence identity” of two sequences as used herein relates tothe number of positions with identical nucleotides or amino acidsdivided by the number of nucleotides or amino acids in the shorter ofthe sequences, when the two sequences are aligned. In particularembodiments, said sequence identity is from 70% to 80%, from 81% to 85%,from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100%.

The terms “peptide-encoding polynucleotide (or nucleic acid)” and“polynucleotide (or nucleic acid) encoding peptide” as used herein referto a nucleotide sequence, which, when expressed in an appropriateenvironment, results in the generation of the relevant peptide sequenceor a derivative or homologue thereof. Such polynucleotides or nucleicacids include the normal sequences encoding the peptide, as well asderivatives and fragments of these nucleic acids capable of expressing apeptide with the required activity. According to one embodiment, thenucleic acid encoding the peptides according to the invention orfragment thereof is a sequence encoding the peptide or fragment thereoforiginating from a mammal or corresponding to a mammalian, mostparticularly a human peptide fragment.

The term “organic compound having a reducing activity” refers in thecontext of this invention to compounds, more in particular amino acidsequences, with a reducing activity for disulfide bonds on proteins. Theterm “immune disorders” or “immune diseases” refers to diseases whereina reaction of the immune system is responsible for or sustains amalfunction or non-physiological situation in an organism. Included inimmune disorders are, inter alia, allergic disorders and autoimmunediseases.

The terms “allergic diseases” or “allergic disorders” as used hereinrefer to diseases characterised by hypersensitivity reactions of theimmune system to specific substances called allergens (such as pollen,stings, drugs, or food). Allergy is the ensemble of signs and symptomsobserved whenever an atopic individual patient encounters an allergen towhich he has been sensitised, which may result in the development ofvarious diseases, in particular respiratory diseases and symptoms suchas bronchial asthma. Various types of classifications exist and mostlyallergic disorders have different names depending upon where in themammalian body it occurs. “Hypersensitivity” is an undesirable(damaging, discomfort-producing and sometimes fatal) reaction producedin an individual upon exposure to an antigen to which it has becomesensitised; “immediate hypersensitivity” depends of the production ofIgE antibodies and is therefore equivalent to allergy.

The terms “autoimmune disease” or “autoimmune disorder” refer todiseases that result from an aberrant immune response of an organismagainst its own cells and tissues due to a failure of the organism torecognise its own constituent parts (down to the sub-molecular level) as“self”. The group of diseases can be divided in two categories,organ-specific and systemic diseases.

An “allergen” is defined as a substance, usually a macromolecule or aproteic composition which elicits the production of IgE antibodies inpredisposed, particularly genetically disposed, individuals (atopics)patients. Similar definitions are presented in Liebers et al. (1996)Clin. Exp. Allergy 26, 494-516.

The term “therapeutically effective amount” refers to an amount of thepeptide of the invention or derivative thereof, which produces thedesired therapeutic or preventive effect in a patient. For example, inreference to a disease or disorder, it is the amount which reduces tosome extent one or more symptoms of the disease or disorder, and moreparticularly returns to normal, either partially or completely, thephysiological or biochemical parameters associated with or causative ofthe disease or disorder. According to one particular embodiment of thepresent invention, the therapeutically effective amount is the amount ofthe peptide of the invention or derivative thereof, which will lead toan improvement or restoration of the normal physiological situation. Forinstance, when used to therapeutically treat a mammal affected by animmune disorder, it is a daily amount peptide/kg body weight of the saidmammal. Alternatively, where the administration is through gene-therapy,the amount of naked DNA or viral vectors is adjusted to ensure the localproduction of the relevant dosage of the peptide of the invention,derivative or homologue thereof.

The term “natural” when referring to a peptide or a sequence hereinrelates to the fact that the sequence is identical to a naturallyoccurring sequence. In contrast therewith the term “artificial” refersto a sequence or peptide which as such does not occur in nature.Optionally, an artificial sequence is obtained from a natural sequenceby limited modifications such as changing one or more amino acids withinthe naturally occurring sequence or by adding amino acids N- orC-terminally of a naturally occurring sequence. Amino acids are referredto herein with their full name, their three-letter abbreviation or theirone letter abbreviation.

Motifs of amino acid sequences are written herein according to theformat of Prosite. The symbol X is used for a position where any aminoacid is accepted. Alternatives are indicated by listing the acceptableamino acids for a given position, between square brackets (‘[ ]’). Forexample: [CST] stands for an amino acid selected from Cys, Ser or Thr.Amino acids which are excluded as alternatives are indicated by listingthem between curly brackets (‘{ }’). For example: {AM} stands for anyamino acid except Ala and Met. The different elements in a motif areseparated from each other by a hyphen - . Repetition of an identicalelement within a motif can be indicated by placing behind that element anumerical value or a numerical range between parentheses. For example:X(2) corresponds to X—X, X(2, 4) corresponds to X—X or X—X—X or X—X—X—X,A(3) corresponds to A-A-A.

The present invention is based upon the finding that a peptide,comprising a T cell epitope and a peptide sequence, having reducingactivity is capable of generating a population of regulatory T cellswhich have a cytotoxic effect on antigen presenting cells.

Accordingly, in its broadest sense, the invention relates to peptideswhich comprise at least one T-cell epitope of an antigen (self ornon-self) with a potential to trigger an immune reaction, coupled to anorganic compound having a reducing activity, such as a thioreductasesequence motif. The T cell epitope and the organic compound areoptionally separated by a linker sequence. In further optionalembodiments the peptide additionally comprises an endosome targetingsequence and/or additional “flanking” sequences.

The peptides of the invention can be schematically represented as A-L-Bor B-L-A, wherein A represents a T-cell epitope of an antigen (self ornon-self) with a potential to trigger an immune reaction, L represents alinker and B represents an organic compound having a reducing activity.

The reducing activity of an organic compound can be assayed for itsability to reduce a sulfhydryl group such as in the insulin solubilityassay described in the examples herein wherein the solubility of insulinis altered upon reduction, or with a fluorescence-labelled insulin. Thereducing organic compound may be coupled at the amino-terminus side ofthe T-cell epitope or at the carboxy-terminus of the T-cell epitope.

Generally the organic compound with reducing activity is a peptidesequence. Peptide fragments with reducing activity are encountered inthioreductases which are small disulfide reducing enzymes includingglutaredoxins, nucleoredoxins, thioredoxins and other thiol/disulfideoxydoreductases (Holmgren (2000) Antioxid Redox Signal 2, 811-820;Jacquot et al. (2002) Biochem Pharm 64, 1065-1069). They aremultifunctional, ubiquitous and found in many prokaryotes andeukaryotes. They exert reducing activity for disulfide bonds on proteins(such as enzymes) through redox active cysteines within conserved activedomain consensus sequences: C—X(2)-C, C—X(2)-S, C—X(2)-T, S—X(2)-C,T-X(2)-C (Fomenko et al. (2003) Biochemistry 42, 11214-11225; Fomenko etal. (2002) Prot. Science 11: 2285-2296), in which X stands for any aminoacid. Such domains are also found in larger proteins such as proteindisulfide isomerase (PDI) and phosphoinositide-specific phospholipase C(Table 1).

TABLE 1 Occurrence of the motif CST-X(2)-CST in representative reducingproteins C-X(2)-S and C-X(2)-S flanking Protein position in secondaryOrganism length the protein structure # Description D. 129 21-25b-CHGS-coil Der p 2 allergen pteronyssinus E. coli K12 115 30-35b-CGFS-a Glutaredoxin E. coli K12 104 14-18 b-CGTS-a arsenate reductaseS. cerevisiae 203 108-112 b-CPYS-a glutaredoxin homologue S. cerevisiae231 136-140 b-CSYS-a glutaredoxin homologue S. cerevisiae 517 62-66b-CLHS-a protein disulfide isomerase H. sapiens 793 72-78 b-CGHS-athioredoxin homologue # b: beta-strand; a: alpha helix

Accordingly, in particular embodiments, peptides according to thepresent invention comprise the thioreductase sequence motif[CST]-X(2)-[CST] wherein at least one of [CST] is Cys; thus the motif iseither [C]-X(2)-[CST] or [CST]-X(2)-[C]. In the present application sucha tetrapeptide will be referred to as “the motif”. In particularembodiments peptides of the invention contain the sequence motif[C]-X(2)-[CS] or [CS]-X(2)-[C]. In more particular embodiments peptidescontain the sequence motif C—X(2)-S, S—X(2)-C or C—X(2)-C.

As explained in detail further on, the peptides of the present inventioncan be made by chemical synthesis, which allows the incorporation ofnon-natural amino acids. Accordingly, in the motif of reducing compoundsaccording to particular embodiments of the present invention, Crepresents either cysteine or another amino acids with a thiol groupsuch as mercaptovaline, homocysteine or other natural or non-naturalamino acids with a thiol function. In order to have reducing activity,the cysteines present in the motif should not occur as part of a cystinedisulfide bridge. Nevertheless, the motif may comprise modifiedcysteines such as methylated cysteine, which is converted into cysteinewith free thiol groups in vivo.

The amino acid X in the [CST]-X(2)-[CST] motif of particular embodimentsof the reducing compounds of the invention can be any natural aminoacid, including S, C, or T or can be a non-natural amino acid. Inparticular embodiments X is an amino acid with a small side chain suchas Gly, Ala, Ser or Thr. In further particular embodiments, X is not anamino acid with a bulky side chain such as Tyr. In further particularembodiments at least one X in the [CST]-X(2)-[CST] motif is His or Pro.

In the peptides of the present invention comprising the motif describedabove as the reducing compound, the motif is located such that, when theepitope fits into the MHC groove, the motif remains outside of the MHCbinding groove. The motif is placed either immediately adjacent to theepitope sequence within the peptide, or is separated from the T cellepitope by a linker. More particularly, the linker comprises an aminoacid sequence of 7 amino acids or less. Most particularly, the linkercomprises 1, 2, 3, or 4 amino acids. Alternatively, a linker maycomprise 6, 8 or 10 amino acids. In those particular embodiments of thepeptides of the invention where the motif sequence is adjacent to theepitope sequence this is indicated as position P−4 to P−1 or P+1 to P+4compared to the epitope sequence.

Apart from a peptide linker other organic compounds can be used aslinker to link the parts of the peptide to each other (e.g. the motif tothe T cell epitope sequence).

The peptides of the present invention can further comprise additionalshort amino acid sequences N or C-terminally of the (artificial)sequence comprising the T cell epitope and the reducing compound(motif). Such an amino acid sequence is generally referred to herein asa ‘flanking sequence’. A flanking sequence can be positioned between theepitope and an endosomal targeting sequence and/or between the reducingcompound (e.g. motif) and an endosomal targeting sequence. In furtherembodiments, not comprising an endosomal targeting sequence, a shortamino acid sequence may be present N and/or C terminally of the reducingcompound and/or epitope sequence in the peptide. More particularly aflanking sequence is a sequence of between 1 and 7 amino acids, mostparticularly a sequence of 2 amino acids.

In particular embodiments of the peptides of the invention, the motif islocated N-terminal from the epitope.

In further particular embodiments, where the motif present in thepeptide contains one cysteine, this cysteine is present in the motif inthe position remote from the epitope, thus the motif occurs asC—X(2)-[ST] or C—X(2)-S N-terminally of the epitope or occurs as[ST]-X(2)-C or S—X(2)-C C-terminally of the epitope.

In certain embodiments of the present invention, peptides are providedcomprising one epitope sequence and a motif sequence. In furtherparticular embodiments, the motif occurs several times (1, 2, 3, 4 oreven more times) in the peptide, for example as repeats of the motifwhich can be spaced from each other by one or more amino acids (e.g.CXXC X CXXC X CXXC), as repeats which are adjacent to each other (CXXCCXXC CXXC) or as repeats which overlap with each other CXXCXXCXXC orCXCCXCCXCC). Alternatively, one or more motifs are provided at both theN and the C terminus of the T cell epitope sequence.

Other variations envisaged for the peptides of the present inventioninclude peptides which contain repeats of a T cell epitope sequencewherein each epitope sequence is preceded and/or followed by the motif(e.g. repeats of “motif-epitope” or repeats of “motif-epitope-motif”).Herein the motifs can all have the same sequence but this is notobligatory. It is noted that repetitive sequences of peptides whichcomprise an epitope which in itself comprises the motif will also resultin a sequence comprising both the ‘epitope’ and a ‘motif’. In suchpeptides, the motif within one epitope sequence functions as a motifoutside a second epitope sequence.

In particular embodiments however, the peptides of the present inventioncomprise only one T cell epitope.

Accordingly, peptides according to the present invention comprise, inaddition to a reducing compound, a T cell epitope derived from anantigen, typically an allergen or an auto-antigen, depending on theapplication. As described below a T cell epitope in a protein sequencecan be identified by functional assays and/or one or more in silicoprediction assays. The amino acids in a T cell epitope sequence arenumbered according to their position in the binding groove of the MHCproteins. In particular embodiments, the T-cell epitope present withinthe peptides of the invention consists of between 8 and 25 amino acids,yet more particularly of between 8 and 16 amino acids, yet mostparticularly consists of 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.In a more particular embodiment, the T cell epitope consists of asequence of 9 amino acids. In a further particular embodiment, theT-cell epitope is an epitope, which is presented to T cells by MHC-classII molecules. In particular embodiments of the present invention, the Tcell epitope sequence is an epitope sequence which fits into the cleftof an MHC II protein, more particularly a nonapeptide fitting into theMHC II cleft.

The T cell epitope of the peptides of the present invention cancorrespond either to a natural epitope sequence of a protein or can be amodified version thereof, provided the modified T cell epitope retainsits ability to bind within the MHC cleft, similar to the natural T cellepitope sequence. The modified T cell epitope can have the same bindingaffinity for the MHC protein as the natural epitope, but can also have alowered affinity. In particular embodiments the binding affinity of themodified peptide is no less than 10-fold less than the original peptide,more particularly no less than 5 times less. It is a finding of thepresent invention that the peptides of the present invention have astabilising effect on protein complexes. Accordingly, the stabilisingeffect of the peptide-MHC complex compensates for the lowered affinityof the modified epitope for the MHC molecule. An example hereof is theIle28Asn substitution of Der p 2 peptide p21-35, which despite a loweraffinity for the MHC II cleft, is capable of eliciting the same T cellresponse as the natural Der p 2 peptide p21-35.

In particular embodiments, the sequence comprising the T cell epitopeand the reducing compound within the peptide is further linked to anamino acid sequence (or another organic compound) that facilitatesuptake of the peptide into late endosomes for processing andpresentation within MHC class II determinants. The late endosometargeting is mediated by signals present in the cytoplasmic tail ofproteins and correspond to well-identified peptide motifs such as thedileucine-based [DE]XXXL[LI] or DXXLL motif (e.g. DXXXLL), thetyrosine-based YXXØ motif or the so called acidic cluster motif. Thesymbol 0 represents amino acid residues with a bulky hydrophobic sidechains such as Phe, Tyr and Trp. The late endosome targeting sequencesallow for processing and efficient presentation of the antigen-derived Tcell epitope by MHC-class II molecules. Such endosomal targetingsequences are contained, for example, within the gp75 protein(Vijayasaradhi et al. (1995) J Cell Biol 130, 807-820), the human CD3gamma protein, the HLA-BM 11 (Copier et al. (1996) J. Immunol. 157,1017-1027), the cytoplasmic tail of the DEC205 receptor (Mahnke et al.(2000) J Cell Biol 151, 673-683). Other examples of peptides whichfunction as sorting signals to the endosome are disclosed in the reviewof Bonifacio and Traub (2003) Annu. Rev. Biochem. 72, 395-447.Alternatively, the sequence can be that of a subdominant or minor T cellepitope from a protein, which facilitates uptake in late endosomewithout overcoming the T cell response towards the antigen, i.e.allergen or auto-antigen derived T cell epitope.

The late endosome targeting sequence can be located either at theamino-terminal or at the carboxy-terminal end of the allergen orautoantigen-derived peptide for efficient uptake and processing and canalso be coupled through a flanking sequence, such as a peptide sequenceof up to 10 amino acids. When using a minor T cell epitope for targetingpurpose, the latter is typically located at the amino-terminal end ofthe allergen or autoantigen derived peptide.

Accordingly, the present invention envisages peptides of antigenicproteins and their use in eliciting specific immune reactions. Thepeptides of the present invention can either correspond to fragments ofproteins which comprise, within their sequence, the features of thepresent invention, i.e. a reducing compound and a T cell epitopeseparated by at most 10, preferably 7 amino acids or less.Alternatively, and for most antigenic proteins, the peptides of theinvention are generated by coupling a reducing compound, moreparticularly a reducing motif as described herein, N-terminally orC-terminally to a T cell epitope of the antigenic protein (eitherdirectly adjacent thereto or with a linker of at most 10, moreparticularly at most 7 amino acids) so as to obtain the characterisingfeatures of the invention. Moreover the T cell epitope sequence of theprotein and/or the motif can be modified and/or one or more flankingsequences and/or a targeting sequence can be introduced (or modified),compared to the naturally occurring sequence. Thus, depending on whetheror not the features of the present invention can be found within thesequence of the antigenic protein of interest, the peptides of thepresent invention can comprise a sequence which is ‘artificial’ or‘naturally occurring’.

The peptides of the present invention can vary substantially in length.In particular embodiments, where the reducing compound corresponds tothe motif as described herein, the length of the peptides vary from12-13 amino acids, i.e. consisting of an epitope of 8-9 amino acids andadjacent thereto the motif as described herein of 4 amino acids, up to50 or more amino acids. For example, a peptide according to theinvention may comprise an endosomal targeting sequence of 40 aminoacids, a flanking sequence of about 2 amino acids, a motif as describedherein of 4 amino acids, a linker of 4 amino acids and a T cell epitopepeptide of 9 amino acids.

Accordingly, in particular embodiments, the complete peptides consist ofbetween 12 amino acids and 20 up to 25, 30, 50, 75, 100 or 200 aminoacids. In a more particular embodiments, the peptides consists ofbetween 10 and 20 amino acids. More particularly, where the reducingcompound is a motif as described herein, the length of the (artificialor natural) sequence comprising the epitope and motif optionallyconnected by a linker (referred to herein as ‘epitope-motif’ sequence),without the endosomal targeting sequence, is critical. The‘epitope-motif’ more particularly has a length of 12, 13, 14, 15, 16,17, 18 or 19 amino acids, optimally of 18 amino acids or less. Suchpeptides of 12 or 13 to 18 amino acids can optionally be coupled to anendosomal targeting signal of which the size is less critical.

As detailed above, in particular embodiments, the peptides of thepresent invention comprise a reducing motif as described herein linkedto a T cell epitope sequence. According to a particular embodiment thepeptides are peptides from proteins which do not comprise within theirnative natural sequence an amino acid sequence with redox properties inthe vicinity (i.e. within a sequence of 11 amino acids N or Cterminally) of the epitope of interest, more specifically which do notcomprise in their native natural sequence a consensus sequence ofthioredoxin, glutaredoxin or thioreductases or homologues thereof, inthe vicinity of the epitope of interest. Most particularly, theinvention encompasses generating immunogenic peptides from antigenicproteins which do not comprise a sequence selected from C—X(2)-S,S—X(2)-C, C—X(2)-C, S—X(2)-S, C—X(2)-T, T-X(2)-C, or any other consensussequences which are typical of consensus sequences of thioredoxin,glutaredoxin or thioreductases in the vicinity of the epitope ofinterest, i.e. within a sequence of 11 amino acids N or C terminally ofthe epitope sequence. In further particular embodiments, the presentinvention provides immunogenic peptides of antigenic proteins which donot comprise the above-described amino acid sequences with redoxproperties within their sequence. In further particular embodiments, theantigenic protein does not naturally comprise the sequence C-H-G-Swithin a sequence of 11 amino acids N or C terminally of the epitopesequence. More particularly the present invention claims peptides otherthan peptides comprising epitope EPCIIHRGKP [SEQ ID. NO: 1] of thep21-35 peptide of Der p 2 (CHGSEPCIIHRGKPF [SEQ ID. NO: 2]) and motifC-H-G-S [SEQ ID. NO: 3], in particularly those peptides where this motifis located N-terminally from the epitope sequence, as such peptidescorrespond to peptides generated in the prior art and used for inducingan immune response in the context of epitope mapping of Der p 2 (Wu etal. 2002, J. Immunol. 169, 2430-2435).

In further particular embodiments, the peptides of the invention arepeptides comprising T cell epitopes which do not comprise an amino acidsequence with redox properties within their natural sequence.

However, in alternative embodiments, the T cell epitope may comprise anysequence of amino acids ensuring the binding of the epitope to the MHCcleft. Where an epitope of interest of an antigenic protein comprises amotif such as described herein within its epitope sequence, theimmunogenic peptides according to the present invention comprise thesequence of a motif as described herein and/or of another reducingsequence coupled N- or C-terminally to the epitope sequence such that(contrary to the motif present within the epitope, which is buriedwithin the cleft) the attached motif can ensure the reducing activity.

In particular embodiments, the peptides of the present invention are notnatural but artificial peptides which contain, in addition to a T cellepitope, a motif as described herein, whereby the motif is optionallyseparated from the T cell epitope by a linker consisting of up to seven,most particularly up to four amino acids.

In a particular embodiment, peptides according to the invention areprovided of the Der p 2 protein, comprising a motif as described hereinand a Der p 2 epitope. More particularly peptides are providedcomprising one or more copies of the nonapeptide epitope EPCIIHRGKP [SEQID. NO: 1] of Der p 2 each linked to a reducing motif consisting of theC—X2-C motif. Alternatively, peptides are provided of the Der p 2protein comprising a T cell epitope other than a sequence comprisingEPCIIHRGKP [SEQ ID. NO: 1] linked to a motif C—X(2)-[CST] or[CST]-X(2)-C. The peptides of the invention optionally comprise anendosomal targeting sequence. Most particularly, immunogenic peptides ofDer p 2 are provided comprising the C—X2-C motif.

Another aspect of the present invention relates to the processes forgenerating immunogenic peptides of the present invention describedherein.

In a first embodiment of methods according to the present invention apeptide of an antigenic protein capable of eliciting cytolytic CD4+ Tcell activity is prepared by providing a peptide consisting of a T-cellepitope of said antigenic protein, and linking to said epitope areducing compound. More particularly methods according to the inventionencompass linking to said T cell epitope a sequence of amino acidscorresponding to the motif C—X(2)-[CST] or [CST]-X(2)-C, such that saidmotif and said epitope are either adjacent to each other or separated bya linker of at most 7 amino acids, more particularly at most 4 aminoacids. More particularly, the motif corresponds to C—X2-C.

In particular embodiments of the methods of the invention, the T cellepitope of the antigenic protein is selected such that the antigenicprotein does not comprise, in its natural sequence, a sequencecorresponding to the combined sequence of the epitope and a motifaccording to the present invention within a region of 11 amino acids N-or C-terminally of said epitope. In further particular embodiments,where the epitope comprises the sequence EPCIIHRGKP [SEQ ID. NO: 1] ofthe p21-35 peptide of Der p 2, the motif corresponds to [CT]-X2-C orC—X2-[CT], most particularly to C—X2-C. The methods of the presentinvention generate immunogenic peptides which induce a specific immuneresponse which is not generated, or not to that extent, by the naturallygenerated T cell epitopes of the antigenic protein. This effect isensured by the specific combination of T cell epitope and reducingcompound, more particularly of T cell epitope and reducing motif.

Accordingly, methods as described above are particularly suited for thegeneration of immunogenic peptides from allergens or auto-antigens fromproteins which do not have a motif such as described herein in theirsequence or wherein a motif as described herein is present completely orpartially within an epitope sequence of interest or wherein a motif ispresent outside, but remote (i.e. more than 4, 7, 10 amino acids) froman epitope sequence of interest.

In particular embodiments of the methods described above, one or morefurther steps are provided whereby a linker is introduced between the Tcell epitope and the reducing compound and/or further sequences areadded (such as a targeting sequence) and/or one or more flankingsequences and/or modifications are introduced into the epitope sequenceof the peptide.

In further embodiments of methods according to the invention ofobtaining a peptide capable of eliciting cytolytic CD4+ T cell activityof an antigenic protein, methods are provided which ensure theidentification of a suitable immunogenic peptide within an antigenicprotein. In these embodiments, the methods of the present inventionencompass determining whether the antigenic protein comprises, withinits natural sequence, a T cell epitope whereby the protein furthercomprises within a region of 11, more particularly within a region of 8amino acids N- or C-terminally of the T cell epitope, a reducing motifas described herein. Accordingly, these embodiments comprise theidentification within the antigenic protein of a suitable sequence foruse as an immunogenic peptide and the production of a peptidecorresponding to the identified sequence. More particularly, theisolated peptides generated in this way comprise a length of between 12and 19 amino acids. Methods for production of peptides are describedbelow. Where suitable enzymatic cleavage sites are present in theprotein, it is further envisaged that the peptides of the presentinvention can also be generated by enzymatic cleavage from the nativeprotein.

In particular embodiments of the different methods described above, oneor more further steps are provided whereby further sequences are addedto the peptides obtained, such as a targeting sequence and/or one ormore flanking sequences and/or modifications are introduced into theepitope, linker and/or reducing motif of the peptide. Thesemodifications may further enhance the immunogenic properties of thepeptide or may improve other characteristics of the peptides such asease of synthesis, solubility, etc.

The methods described above will allow the generation of an immunogenicpeptide according to the present invention only for a selection ofantigenic proteins which naturally comprise, in the vicinity of anepitope of interest, a C—X(2)-[CST] or [CST]-X(2)-C motif. In particularembodiments the T cell epitope does not comprise sequence EPCIIHRGKP[SEQ ID. NO: 1] of the p21-35 peptide of Der p 2. In particularembodiments, the antigenic protein may naturally comprise a C—X(2)-[ST]or [ST]-X(2)-C motif within a sequence of maximally 11 amino acidsflanking the epitope of interest, and the methods of the inventiongenerating an isolated peptide comprising said motif and said epitopesequence and modifying said motif to C—X(2)-C so as to further increasethe immunogenic properties described herein.

The identification of a suitable T cell epitope of an antigenic proteinfor use in the generation of peptides as described in the methods aboveis detailed below.

It has been shown that upon administration (i.e. injection) to a mammalof a peptide according to the invention (or a composition comprisingsuch a peptide), the peptide elicits the activation of T cellsrecognising the antigen (i.e. the allergen or auto-antigen) derived Tcell epitope and provides an additional signal to the T cell throughreduction of surface receptor. This supra-optimal activation results inT cells acquiring cytotoxic properties for the cell presenting the Tcell epitope, as well as suppressive properties on bystander T cells. Inthis way, the peptides or composition comprising the peptides describedin the present invention, which contain an antigen derived T cellepitope and, outside the epitope, a reducing compound can be used fordirect immunisation of mammals, including human beings.

One aspect of the invention thus provides peptides of the invention orderivatives thereof, for use as a medicine. Accordingly, the presentinvention provides therapeutic methods which comprise administering oneor more peptides according to the present invention to a patient in needthereof.

The present invention offers methods by which allergen/antigen-specificT cells endowed with cytotoxic properties can be elicited byimmunisation with small peptides. It has been found that peptides whichcontain (i) a sequence encoding a T cell epitope from an antigen (i.e.allergen, auto-antigen) and (ii) a consensus sequence with redoxproperties, and further optionally also comprising a sequence tofacilitate the uptake of the peptide into late endosomes for efficientMHC-class II presentation, elicit suppressor T-cells.

The immunogenic properties of the peptides of the present invention areof particular interest in the treatment and prevention of immunereactions. Accordingly, another aspect of the present invention providesfor the use of the peptides described herein as a medicament, moreparticularly for the manufacture of a medicament for the prevention ortreatment of an immune disorder in a mammal, more in particular in ahuman.

Another aspect of the present invention thus relates to a method oftreatment or prevention of an immune disorder of a mammal in need forsuch treatment or prevention, by using the peptides of the invention,homologues or derivatives thereof, the methods comprising the step ofadministering to said mammal suffering or at risk of an immune disordera therapeutically effective amount of the peptides of the invention,homologues or derivatives thereof such as to reduce the symptoms of theimmune disorder. The treatment of both humans and animals, such as, butnot limited to pets and horses is envisaged.

The immune disorders referred to above are in a particular embodimentselected from allergic diseases and autoimmune diseases. Allergicdiseases are conventionally described as type-1 mediated diseases orIgE-mediated diseases. Clinical manifestations of allergic diseasesinclude bronchial asthma, allergic rhinitis, atopic dermatitis, foodhypersensitivity and anaphylactic reactions to insect bites or drugs.Allergic diseases are caused by hypersensitivity reactions of the immunesystem to specific substances called allergens (such as pollen, stings,drugs, or food). The most severe form of an allergic disorder isanaphylactic shock, which is a medical emergency. Allergens includeairborne allergens, such as those of house dust mite, pets and pollens.Allergens also include ingested allergens responsible for foodhypersensitivity, including fruits, vegetables and milk.

In order to treat the above diseases, peptides according to theinvention are generated from the antigenic proteins or allergens knownor believed to be a causative factor of the disease. The allergens thatcan be used for selection of T-cell epitopes are typically allergenswhich are selected from the group consisting of:

-   -   food allergens present in peanuts, fish e.g. codfish, egg white,        crustacea e.g. shrimp, milk e.g. cow's milk, wheat, cereals,        fruits of the Rosacea family (apple, plum, strawberry),        vegetables of the Liliacea, Cruciferae, Solanaceae and        Umbelliferae families, tree nuts, sesame, peanut, soybean and        other legume family allergens, spices, melon, avocado, mango,        fig, banana, . . . .    -   house dust mites allergens obtained from Dermatophagoides spp        or D. pteronyssinus, D. farinae and D. microceras, Euroglyphus        maynei or Blomia sp.,    -   allergens from insects present in cockroach or Hymenoptera,    -   allergens from pollen, especially pollens of tree, grass and        weed,    -   allergens from animals, especially in cat, dog, horse and        rodent,    -   allergens from fungi, especially from Aspergillus, Alternaria or        Cladosporium, and    -   occupational allergens present in products such as latex,        amylase, etc.

The T cell epitope corresponding to an antigenic protein (or immunogen)suitable for use in the context of the present invention is typically auniversal or promiscuous T cell epitope (i.e. a T cell epitope capableof binding to a majority of the MHC class II molecules), moreparticularly present upon an airborne allergen or a foodborne allergen.In particular embodiments, said allergen is selected from the groupconsisting of rhino-sinusitis allergens, allergic bronchial asthmaallergens and atopic dermatitis allergens.

Allergens can also be main allergens present in moulds or various drugssuch as hormones, antibiotics, enzymes, etc. (See also the definition inClin. Exp. Allergy 26, 494-516 (1996) and in Molecular Biology ofAllergy and Immunology, Ed. R. Bush (1996)). Other allergens related tospecific allergic diseases are also well known in the art and can befound on the internet, e.g. on www.allergome.org.

Autoimmune diseases are broadly classified into two categories,organ-specific and systemic diseases. The precise aetiology of systemicauto-immune diseases is not identified. In contrast, organ-specificauto-immune diseases are related to a specific immune response includingB and T cells, which targets the organ and thereby induces and maintainsa chronic state of local inflammation. Examples of organ-specificauto-immune diseases include type 1 diabetes, myasthenia gravis,thyroiditis and multiple sclerosis. In each of these conditions, asingle or a small number of auto-antigens have been identified,including insulin, the acetylcholine muscle receptor, thyroid peroxidaseand major basic protein, respectively. It is well recognised thatsuppression of this organ-specific immune response is beneficial andleads to partial or complete recovery of organ function. There is,however, no therapy, which would suppress such an immune response in anantigen-specific manner. Current therapy rather makes use ofnon-specific suppression obtained by the use of corticosteroids andimmunosuppressive agents, all exhibiting significant side-effectsrelated to their absence of specificity, thereby limiting their use andtheir overall efficacy. Table 2 shows a non-limiting list of examples ofknown auto-antigens which are linked to organ specific autoimmunedisorders and which are envisaged within the context of the presentinvention.

TABLE 2 Representative auto-antigens and diseases linked therewithDisease antigen thyroid diseases thyroglobulin thyroid peroxidase TSHreceptor type 1 diabetes insulin (proinsulin) glutamic aciddecarboxylase (GAD) tyrosine phosphatase IA-2 heat-shock protein HSP65islet-specific glucose6-phosphatase catalytic subunit related protein(IGRP) adrenalitis 21-OH hydroxylase polyendocrine syndromes 17-alphahydroxylase histidine decarboxylase tryptophan hydroxylase tyrosinehydroxylase gastritis & pernicious H+/K+ ATPase intrinsic factor anemiamultiple sclerosis myelin oligodendrocyte glycoprotein (MOG) myelinbasic protein (MBP) proteolipid protein (PLP) myasthenia gravisacetyl-choline receptor ocular diseases retinol-binding protein (RBP)inner ear diseases type II and type IX collagen celiac disease tissuetransglutaminase inflammatory bowel pANCA histone H1 protein diseasesatherosclerosis heat-shock protein HSP60

According to the present invention, immunogenic peptides are providedwhich comprise a T-cell epitope of an antigen (self or non-self) with apotential to trigger an immune reaction, such as an allergen or anauto-antigen, such as those described in Table 2. In a particularembodiment, the T-cell epitope is a dominant T-cell epitope.

Accordingly, in particular embodiments, the methods of treatment andprevention of the present invention comprise the administration of animmunogenic peptide as described herein, wherein the peptide comprise aT-cell epitope of an antigenic protein which plays a role in the diseaseto be treated (for instance such as those described in Table 2 above).In further particular embodiments, the epitope used is a dominantepitope.

The identification and selection of a T-cell epitope from such antigenicproteins, more in particular from allergens or auto-antigens, for use inthe context of the present invention is known to a person skilled in theart.

To identify an epitope suitable for use in the context of the presentinvention, isolated peptide sequences of an antigenic protein are testedby, for example, T cell biology techniques, to determine whether thepeptide sequences elicit a T cell response. Those peptide sequencesfound to elicit a T cell response are defined as having T cellstimulating activity.

Human T cell stimulating activity can further be tested by culturing Tcells obtained from an individual sensitive to e.g. a mite allergen,(i.e. an individual who has an IgE mediated immune response to a miteallergen) with a peptide/epitope derived from the allergen anddetermining whether proliferation of T cells occurs in response to thepeptide/epitope as measured, e.g., by cellular uptake of tritiatedthymidine. Stimulation indices for responses by T cells topeptides/epitopes can be calculated as the maximum CPM in response to apeptide/epitope divided by the control CPM. A T cell stimulation index(S.I.) equal to or greater than two times the background level isconsidered “positive.” Positive results are used to calculate the meanstimulation index for each peptide/epitope for the group ofpeptides/epitopes tested.

Non-natural (or modified) T-cell epitopes can further optionally betested on their binding affinity to MHC class II molecules. This can beperformed in different ways. For instance, soluble HLA class IImolecules are obtained by lysis of cells homozygous for a given class IImolecule. The latter is purified by affinity chromatography. Solubleclass II molecules are incubated with a biotin-labelled referencepeptide produced according to its strong binding affinity for that classII molecule. Peptides to be assessed for class II binding are thenincubated at different concentrations and their capacity to displace thereference peptide from its class II binding is calculated by addition ofneutravidin. Methods can be found in for instance Texier et al., (2000)J. Immunology 164, 3177-3184.)

According to the present invention, the immunogenic properties of T cellepitopes is increased by linking it to a reducing compound.Particularly, peptides of the present invention comprising at least oneT cell epitope and the reducing compound as described herein have a meanT cell stimulation index of greater than or equal to 2.0. A peptidehaving a T cell stimulation index of greater than or equal to 2.0 isconsidered useful as a therapeutic agent. More particularly, peptidesaccording to the invention have a mean T cell stimulation index of atleast 2.5, at least 3.5, at least 4.0, or even at least 5.0. Inaddition, peptides have typically a positivity index (P.I.) of at leastabout 100, at least 150, at least about 200 or at least about 250. Thepositivity index for a peptide is determined by multiplying the mean Tcell stimulation index by the percent of individuals, in a population ofindividuals sensitive to house dust mite (e.g., at least 9 individuals,at least 16 individuals or at least 29 or 30, or even more), who have Tcells that respond to the peptide (thus corresponding to the SImultiplied by the promiscuous nature of the peptide/epitope). Thus, thepositivity index represents both the strength of a T cell response to apeptide (S.I.) and the frequency of a T cell response to a peptide in apopulation of individuals sensitive to house dust mite.

In order to determine optimal T cell epitopes by, for example, finemapping techniques, a peptide having T cell stimulating activity andthus comprising at least one T cell epitope as determined by T cellbiology techniques is modified by addition or deletion of amino acidresidues at either the amino- or carboxyterminus of the peptide andtested to determine a change in T cell reactivity to the modifiedpeptide. If two or more peptides which share an area of overlap in thenative protein sequence are found to have human T cell stimulatingactivity, as determined by T cell biology techniques, additionalpeptides can be produced comprising all or a portion of such peptidesand these additional peptides can be tested by a similar procedure.Following this technique, peptides are selected and producedrecombinantly or synthetically. T cell epitopes or peptides are selectedbased on various factors, including the strength of the T cell responseto the peptide/epitope (e.g., stimulation index) and the frequency ofthe T cell response to the peptide in a population of individuals.

Additionally and/or alternatively, one or more in vitro algorithms canbe used to identify a T cell epitope sequence within an antigenicprotein. Suitable algorithms include, but are not limited to those foundon the following websites:

-   -   http://antigen.i2r.a-star.edu.sg/predBalbc/;    -   http://antigen.i2r.a-star.edu.sq/predBalbc/;    -   http://www.imtech.res.in/raghava/mhcbn;    -   http://www.syfpeithi.de/home.htm;    -   http://www-bs.informatik.uni-tuebingen.de/SVMHC;    -   http://bio.dfci.harvard.edu/Tools/antigenic.html;    -   http://www.jenner.ac.uk/MHCPred/.        More particularly, such algorithms allow the prediction within        an antigenic protein of one or more nonapeptide sequences which        will fit into the groove of an MHC II molecule.

The peptides of the present invention can be generated using recombinantDNA techniques, in bacteria, yeast, insect cells, plant cells ormammalian cells. In view of the limited length of the peptides, they canbe prepared by chemical peptide synthesis, wherein peptides are preparedby coupling the different amino acids to each other. Chemical synthesisis particularly suitable for the inclusion of e.g. D-amino acids, aminoacids with non-naturally occurring side chains or natural amino acidswith modified side chains such as methylated cysteine.

Chemical peptide synthesis methods are well described and peptides canbe ordered from companies such as Applied Biosystems and othercompanies.

Peptide synthesis can be performed as either solid phase peptidesynthesis (SPPS) or contrary to solution phase peptide synthesis. Thebest-known SPPS methods are t-Boc and Fmoc solid phase chemistry:

During peptide synthesis several protecting groups are used. For examplehydroxyl and carboxyl functionalities are protected by t-butyl group,lysine and tryptophan are protected by t-Boc group, and asparagines,glutamine, cysteine and histidine are protected by trityl group, andarginine is protected by the pbf group. In particular embodiments, suchprotecting groups can be left on the peptide after synthesis.

Peptides can be linked to each other to form longer peptides using aligation strategy (chemoselective coupling of two unprotected peptidefragments) as originally described by Kent (Schnolzer & Kent (1992) Int.J. Pept. Protein Res. 40, 180-193) and reviewed for example in Tam etal. (2001) Biopolymers 60, 194-205 provides the tremendous potential toachieve protein synthesis which is beyond the scope of SPPS. Manyproteins with the size of 100-300 residues have been synthesisedsuccessfully by this method. Synthetic peptides have continued to playan ever increasing crucial role in the research fields of biochemistry,pharmacology, neurobiology, enzymology and molecular biology because ofthe enormous advances in the SPPS.

Alternatively, the peptides can be synthesised by using nucleic acidmolecules which encode the peptides of this invention in an appropriateexpression vector which include the encoding nucleotide sequences. SuchDNA molecules may be readily prepared using an automated DNA synthesiserand the well-known codon-amino acid relationship of the genetic code.Such a DNA molecule also may be obtained as genomic DNA or as cDNA usingoligonucleotide probes and conventional hybridisation methodologies.Such DNA molecules may be incorporated into expression vectors,including plasmids, which are adapted for the expression of the DNA andproduction of the polypeptide in a suitable host such as bacterium, e.g.Escherichia coli, yeast cell, animal cell or plant cell.

The physical and chemical properties of a peptide of interest (e.g.solubility, stability) is examined to determine whether the peptideis/would be suitable for use in therapeutic compositions. Typically thisis optimised by adjusting the sequence of the peptide. Optionally, thepeptide can be modified after synthesis (chemical modifications e.g.adding/deleting functional groups) using techniques known in the art.

T cell epitopes on their own are thought to trigger early events at thelevel of the T helper cell by binding to an appropriate HLA molecule onthe surface of an antigen presenting cell and stimulating the relevant Tcell subpopulation. These events lead to T cell proliferation,lymphokine secretion, local inflammatory reactions, the recruitment ofadditional immune cells to the site, and activation of the B cellcascade leading to production of antibodies. One isotype of theseantibodies, IgE, is fundamentally important in the development ofallergic symptoms and its production is influenced early in the cascadeof events, at the level of the T helper cell, by the nature of thelymphokines secreted. A T cell epitope is the basic element or smallestunit of recognition by a T cell receptor where the epitope comprisesamino acid residues essential to receptor recognition, which arecontiguous in the amino acid sequence of the protein.

However, upon administration of the peptides according to the invention(which comprise a T-cell epitope coupled to a redox sequence) orcompositions thereof, the following events are believed to happen:

-   -   activation of antigen (i.e. allergen or auto-antigen) specific T        cells resulting from cognate interaction with the antigen (i.e.        allergen or auto-antigen) derived peptide presented by MHC-class        II molecules;    -   the reductase consensus sequence reduces T cell surface        proteins, such as the CD4 molecule (and also CD3), the second        domain of which contains a constrained disulfide bridge. This        transduces a signal into T cells. Among a series of consequences        related to increased oxidative pathway, important events are        increased calcium influx and translocation of the NF-kB        transcription factor to the nucleus. The latter results in        increased transcription of IFN-gamma and granzymes, which allows        cells to acquire cytotoxic properties;    -   the cytotoxicity affects cells presenting the peptide by a        mechanism, which involves granzyme B secretion, and Fas-FasL        interactions. Destruction of the antigen-presenting target cells        prevents activation of other T cells specific for epitopes        located on the same antigen, or to an unrelated antigen that        would be processed by the same antigen-presenting cell;    -   an additional consequence of T cell activation is to suppress        activation of bystander T cells by a cell-cell contact dependent        mechanism. In such a case, T cells activated by an antigen        presented by a different antigen-presenting cell is also        suppressed provided both cytotoxic and bystander T cells are in        close proximity.

The above-postulated mechanism of action is substantiated withexperimental data (see examples below). Some experiments have alsosuggested, in addition, the involvement of the perforin pathway and/orthe activation of indoleamine oxidase in target cells, which results inincreased tryptophan catabolism, an amino acid essential for cellsurvival, as well as the production of microparticles by activatedTregs.

Experiments in in vivo models described below have demonstrated thatadministration (i.e. injection) of a peptide according to the inventionor a composition thereof can prevent or suppress an antigen-specificimmune response. As an example, in a mouse model of asthma due tosensitisation to an allergen, Der p 2, pre-immunisation with a peptideaccording to the invention described above prevents lung inflammatorycell infiltration, which is a hallmark of asthma. Non-specific airwayhyperreactivity, as measured by inhalation of increasing dosage ofmethacholine, is essentially prevented by the same experimentalprotocol. Migration of cells into the broncho-alveolar fluid is alsofully prevented (see example 4). Furthermore, adoptive transfer of Tcell clones (derived from animals immunised with the composition) fullyprevents and suppresses both the infiltration of inflammatory cells andairway hyperreactivity in recipients. Such T cell clones which areelicited by the administration of the peptides according to theinvention present a number of phenotypic characteristics, which makethem distinct from currently identified Tregs, either natural Tregs oradaptive Tregs. Thus, cytotoxic Tregs show increased expression ofsurface markers including CD103, CTLA-4, FasL and ICOS (uponactivation). They are CD25high even at rest and CD127low (IL7-R).Transcription factor expression includes T-bet but not the transcriptionrepressor Foxp3, considered as a hallmark of natural Tregs. Apart fromhigh production of IFN-gamma, such clones secrete no or only traceamounts of IL-10, IL-4, IL-5, IL-13 or TGF-beta. The abovecharacteristics are not exclusive, but provided as an illustration ofthe fact that such cytotoxic Tregs are distinguishable from other knownTregs.

Accordingly, in yet a further aspect, the present invention providesmethods for generating antigen-specific cytotoxic Tregs either in vivoor in vitro and, independently thereof, methods to discriminatecytotoxic Tregs from other Tregs based on the above-describedcharacteristic expression data.

One aspect of the present invention relates to in vivo methods for theproduction of the antigen-specific Tregs of the invention. A particularembodiment relates to the method for producing or isolating said Tregsby immunising animals (including humans) with the peptides of theinvention as described herein and than isolating the Tregs from saidimmunised animals. More detailed procedures are described in theExamples section herein and are part of the invention.

A further aspect of the present invention relates to in vitro methodsfor the production of antigen-specific Tregs of the invention. Tregs arecrucial in immunoregulation and have great therapeutic potential. Theefficacy of Treg-based immunotherapy critically depends on the Agspecificity of the regulatory T cells. Moreover, the use of Ag-specificTreg as opposed to polyclonal expanded Treg reduces the total number ofTreg necessary for therapy. The generation of regulatory T cells invitro using methods known in the art has a number of importantdisadvantages. The percentage of Tregs within a sample of isolatedperipheral cells is about 5% and the cultivation of Treg cells isdifficult, such that the amount of Tregs is always limited. Moreover,the Tregs generated in this way are non-specific and thus are notcapable of suppressing a specific immune response, but have only generalimmunosuppressive effect when administered to a patient. Accordingly,strategies have been developed for the development of selectionprocedures that allow selection and expansion of highly potent,Ag-specific Tregs. However, the number of antigen-specific Treg cellsobtained in this way remains limited.

The present invention provides methods for generating antigen-specificregulatory T cells having cytotoxic activity.

In one embodiment, methods are provided which comprise the isolation ofperipheral blood cells, the stimulation of the cell population in vitroby an immunogenic peptide according to the invention and the expansionof the stimulated cell population, more particularly in the presence ofIL-2. The methods according to the invention have the advantage thathigher numbers of Tregs are produced and that the Tregs can be generatedwhich are specific for the antigenic protein (by using a peptidecomprising an antigen-specific epitope).

In an alternative embodiment, the Tregs can be generated in vivo, i.e.by the injection of the immunogenic peptides described herein to asubject, and collection of the Tregs generated in vivo.

The antigen-specific regulatory T cells obtainable by the methods of thepresent invention are of particular interest for the administration tomammals for immunotherapy, in the prevention of allergic reactions andthe treatment of relapses in auto-immune diseases. Both the use ofallogenic and autogeneic cells are envisaged.

Accordingly, one aspect of the present invention provides cytotoxic Tregpopulations characterised as described hereinbelow. More particularly,the populations of Treg populations of the present invention areobtained by the methods described herein.

Accordingly, the present invention provides antigen-specific Tregs withcytotoxic properties according to the invention for use as a medicament,more particularly for use in adoptive cell therapy, more particularly inthe treatment of acute allergic reactions and relapses of autoimmunediseases such as multiple sclerosis. The present invention also relatesto the use of said isolated Tregs or Treg cell populations generated asdescribed herein, more particularly antigen-specific Treg cellpopulations generated as described herein for the manufacture of amedicament for the prevention or treatment of immune disorders.Similarly, the invention relates to methods of treatment by using saidisolated Tregs or generated Treg population.

A further aspect of the present invention provides methods fordiscriminating cytotoxic Treg cells from other Treg cells based onexpression characteristics of the cells. More particularly, methodsaccording to the invention comprise determining whether the Treg cellpopulation demonstrates one or more of the following characteristicscompared to a non-cytotoxic Treg cell population:

-   -   an increased expression of surface markers including CD103,        CTLA-4, FasL and ICOS upon activation,    -   high expression of CD25,    -   expression of CD4, ICOS, CTLA-4, GITR and low or no expression        of CD127 (IL7-R),    -   expression of transcription factor T-bet and egr-2 (Krox-20) but        not of the transcription repressor Foxp3,    -   a high production of IFN-gamma and no or only trace amounts of        IL-10, IL-4, IL-5, IL-13 or TGF-beta.

More particularly, the methods of the present invention comprisedetermining that the cells express CD4, that they do not express IL-10or TGF-beta, that they express Krox-20 and produce granzymes and Fasligand. Most particularly these cells are further selected functionallyas cells that do not respond to the activation by TCR recognition. Infurther particular embodiments, the methods encompass determining all ofthe characteristics described above.

Over recent years much progress has been made in the characterisation ofregulatory T cells (Tregs) both in physiological and pathologicalconditions. More particularly, the potential of using Tregs for thetherapy of some diseases has been discussed. The present invention dealswith the development of a newly defined subset of antigen-specificadoptive Tregs that differs from previous reported Tregs by the methodused for in vitro or in vivo induction and by specific properties. Tregsbelong to two broad categories, i.e. natural Tregs and induced (oradaptive) Tregs. Natural Tregs have first been described in the mouse in1995, and defined as a subset of CD4+ T cells actively selected in thethymus. Such cells are characterised by expression of a number ofsurface markers, including CD25 on resting cells, GITR, CTLA-4 andLAG-3. More recently, natural Tregs have been further defined by thelack of expression of CD127 (IL-7R). The Foxp3 transcription repressorplays a determining role in the selection of natural Tregs. Mutations ofFoxp3 result in the absence of natural Tregs, with an X-linked immunederegulation with polyendocrinopathy, enteropathy, atopic manifestationsand lethal infections. Such natural Tregs suppress various inflammatoryprocesses including gastro-intestinal syndromes. At the molecular level,Foxp3 combines with the NFAT transcription factor in competition withAP1, and thereby regulates the transcription of a number of cytokines.The mechanism of action of natural Tregs is under intense scrutiny. Invitro, such cells produce IL-10 and TGF-beta. In vivo, however,neutralisation of IL-10 and/or TGF-b does not overcome the suppression,indicating that other mechanisms are at play. In vitro, natural Tregssuppress an adaptive response in a cell contact dependent manner.Interestingly, natural Tregs express granzyme proteases such asgranzyme-A (GZ-A) and granzyme-B (GZ-B). Although still controversial, afurther or alternative mechanism of action for natural Tregs seems torely on their capacity to lyse target cells by exocytosis of granzymes.GZ-B deficient Tregs loose partly their capacity to suppress immuneresponse. Adaptive Tregs constitute a heterogeneous family of T cellswhich have in common to be antigen-specific, to exert a suppressiveactivity on bystander T cells and to be induced in the periphery. Th3cells are mainly produced by oral administration of antigen and arefound in mesenteric lymph nodes. Such cells exert their suppressiveactivity by producing high levels of TGF-beta with variable amounts ofIL-4 and IL-10. Tr1 cells produce high concentrations of IL-10 andvarying amounts of TGF-beta. These are induced in vitro by exposure ofnaïve CD4+ T cells to high concentrations of IL-10 or combinedactivation by anti-CD3 and anti-CD46 antibodies. The preciserelationship between Th3 and Tr1 cells is not established, in theabsence of specific phenotypic markers. Not only an overlap betweenthese two adaptive Tregs seems to exist, but also additional subsets arelikely to be defined in forthcoming years. Adaptive Tregs do not expressthe Foxp3 repressor factor. Apart from the production of suppressivecytokines such as IL-10 and/or TGF-beta, it has been reported thatCD4+CD25(−) T cells can be induced to express granzymes, mostly GZ-B, byanti-CD3 and anti-CD46 antibody stimulation. It is not clear whetherthese non-specific, in vitro induced Tregs exert a cytotoxic activitydue to granzyme secretion. The peptides of the invention will, uponadministration to a living animal, typically a human being, elicitspecific T cells exerting a suppressive activity on bystander T cells.The peptides apparently activate the oxidative metabolism of T cellsafter cognate interaction and reduce the constrained disulfide bridge ofthe second extracellular domain of the CD4 molecule.

This mechanism also implies and the experimental results show that thepeptides of the invention, although comprising a specific T-cell epitopeof a certain antigen, can be used for the prevention or treatment ofdisorders elicited by an immune reaction against other T-cell epitopesof the same antigen or in certain circumstances even for the treatmentof disorders elicited by an immune reaction against other T-cellepitopes of other different antigens if they would be presented throughthe same mechanism by MHC class II molecules in the vicinity of T cellsactivated by peptides of the invention.

A further particular aspect of the present invention thus relates to acell type, being T cells, more in particular Tregs or T suppressorcells, characterised in that they express CD4, that they do not expressIL-10 or TGF-beta (whilst other adaptive T cells produce IL-10 and/orTGF beta), that they express Krox-20 and produce granzymes and Fasligand. Most particularly these cells are further selected functionallyas cells that do not respond to the activation by TCR recognition. Moreparticularly, populations of the Treg cell type having thecharacteristics described herein are provided herein, whereby theanergic response is antigen-specific.

In further particular embodiments, the T reg cells of the invention arecharacterised in that they have:

-   -   expression of CD25, CD4, ICOS, CTLA-4, GITR, and no expression        of CD127 (IL7-R), expression of transcription factor T-bet and        egr-2 (Krox-20) but not of Foxp3,    -   an increased expression of markers including FasL and granzymes        (B and C) upon activation,    -   a high production of IFN-gamma.

In a further particular embodiment the invention provides a cell type,being T cells, more in particular Tregs or T suppressor cells,characterised in that they have:

-   -   expression of CD25, CD4, ICOS, CTLA-4, GITR, and no expression        of CD127 (IL7-R), expression of transcription factor T-bet and        egr-2 (Krox-20) but not of Foxp3,    -   an increased expression of markers including FasL and granzymes        (B and C) upon activation,    -   a high production of IFN-gamma.

Most particularly the Treg cells or cell populations of the inventionare characterised in that they have:

-   -   an increased expression of surface markers including CD103,        CTLA-4, FasL and ICOS upon activation,    -   high expression of CD25, whilst other adaptive T cells are CD25        negative, expression of CD4, ICOS, CTLA-4, GITR and low or no        expression of CD127 (IL7-R),    -   expression of transcription factor T-bet and egr-2 (Krox-20) but        not of the transcription repressor Foxp3, whilst other adaptive        T cells are Foxp3 positive,    -   a high production of IFN-gamma and no or only trace amounts of        IL-10, IL-4, IL-5, IL-13 or TGF-beta,    -   an increased expression of markers including FasL and granzymes        (B and C) upon activation.

More particularly, the present invention provides isolated cellpopulations of the cell type having the characteristics described above,which, in addition are antigen-specific, i.e. capable of suppressing anantigen-specific immune response. Accordingly, the present inventionprovides isolated antigen-specific Treg cells, characterised asdescribed above. More particularly the present invention providesantigen specific T-reg cells other than those elicited by Der p 2.

The peptides, according to the invention may also be used in genetherapy methods well known in the art and the terminology used hereinexplaining the use of peptides according to the invention also includesthe use of nucleic acids encoding or expressing immunogenic peptidesaccording to the invention.

Accordingly, a further aspect of the present invention relates tonucleic acid sequences encoding the peptides of the present inventionand methods for their use.

Different methods of achieving, by way of gene therapy, levels ofpeptides, homologues or derivatives thereof according to the inventionin a mammal in vivo are envisaged within the context of the presentinvention.

Recombinant nucleic acid molecules encoding protein sequences can beused as naked DNA or in liposomes or other lipid systems for delivery totarget cells. Other methods for the direct transfer of plasmid DNA intocells are well known to those skilled in the art for use in human genetherapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins. In its simplest form, genetransfer can be performed by simply injecting minute amounts of DNA intothe nucleus of a cell, through a process of microinjection. Oncerecombinant genes are introduced into a cell, they can be recognised bythe cells normal mechanisms for transcription and translation, and agene product will be expressed. Other methods have also been attemptedfor introducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with calcium phosphate andtaken into cells by pinocytosis; electroporation, wherein cells areexposed to large voltage pulses to introduce holes into the membrane);lipofection/liposome fusion, wherein DNA is packed into lipophilicvesicles which fuse with a target cell; and particle bombardment usingDNA bound to small projectiles. Another method for introducing DNA intocells is to couple the DNA to chemically modified proteins. Adenovirusproteins are capable of destabilising endosomes and enhancing the uptakeof DNA into cells. Mixing adenovirus to solutions containing DNAcomplexes, or the binding of DNA to polylysine covalently attached toadenovirus using protein crosslinking agents substantially improves theuptake and expression of the recombinant gene. Adeno-associated virusvectors may also be used for gene delivery into vascular cells. As usedherein, “gene transfer” means the process of introducing a foreignnucleic acid molecule into a cell, which is commonly performed to enablethe expression of a particular product encoded by the gene. The saidproduct may include a protein, polypeptide, anti-sense DNA or RNA, orenzymatically active RNA. Gene transfer can be performed in culturedcells or by direct administration into mammals.

In another embodiment, a vector comprising a nucleic acid moleculesequence encoding a peptide according to the invention is provided. Inparticular embodiments, the vector is generated such that the nucleicacid molecule sequence is expressed only in a specific tissue. Methodsof achieving tissue-specific gene expression are well known in the art.According to one embodiment, this is achieved by placing the sequenceencoding a peptide according to the invention under control of apromoter which directs expression in one or more particular tissues.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adenovirus, adeno-associated virus, herpes viruses, RNA virusesor bovine papilloma virus, may be used for delivery of nucleotidesequences (e.g., cDNA) encoding peptides, homologues or derivativesthereof according to the invention into the targeted tissues or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant viral vectors containing such codingsequences.

Accordingly, the present invention discloses the use of a nucleic acidwhich is capable of expressing the peptides of the invention, in vivo,for the treatment and/or prevention of allergic and autoimmune diseases.According to one embodiment, the nucleic acid capable of expressing apeptide according to the invention in vivo is a sequence encoding such apeptide, which is operably linked to a promoter. Such a sequence can beadministered directly or indirectly. For instance, an expression vectorcontaining the coding sequence for a peptide according to the inventionmay be inserted into cells, after which the said cells are grown invitro and then injected or infused into the patient. Alternatively thenucleic acid capable of expressing a peptide according to the inventionin vivo is a sequence which modifies endogenous expression of the cells.The gene therapy method may involve the use of an adenovirus vectorincluding a nucleotide sequence coding for peptides, homologues orderivatives thereof according to the invention or a naked nucleic acidmolecule coding for a peptide according to the invention. Alternatively,engineered cells containing a nucleic acid molecule coding for a peptideaccording to the invention may be injected.

Where the administration of one or more peptides according to theinvention is ensured through gene transfer (i.e. the administration of anucleic acid which ensures expression of peptides according to theinvention in vivo upon administration), the appropriate dosage of thenucleic acid can be determined based on the amount of peptide expressedas a result of the nucleic acid, such as e.g. by determining theconcentration of peptide in the blood after administration. Thus, in aparticular embodiment, the peptides of the invention are administeredthrough the use of polynucleotides encoding said peptides, whether in anexpression vector or not and thus the present invention also relates togene therapy methods. Another particular embodiment relates to the useof methods to induce a local overexpression of the peptides of theinvention for the treatment or prevention of immune disorders.

Yet another aspect of the present invention provides pharmaceuticalcompositions comprising one or more peptides according to the presentinvention, further comprising a pharmaceutically acceptable carrier. Asdetailed above, the present invention also relates to the compositionsfor use as a medicine or to methods of treating a mammal of an immunedisorder by using said composition and to the use of said compositionsfor the manufacture of a medicament for the prevention or treatment ofimmune disorders.

The pharmaceutical composition could for example be a vaccine suitablefor treating or preventing immune disorders, especially airborne andfoodborne allergy, as well as diseases of allergic origin. As an exampledescribed further herein of a pharmaceutical composition, a peptideaccording to the invention is adsorbed on an adjuvant suitable foradministration to mammals, such as aluminium hydroxide (alum).Typically, 50 μg of the peptide adsorbed on alum are injected by thesubcutaneous route on 3 occasions at an interval of 2 weeks. It shouldbe obvious for those skilled in the art that other routes ofadministration are possible, including oral, intranasal orintramuscular. Also, the number of injections and the amount injectedcan vary depending on the conditions to be treated. Further, otheradjuvants than alum can be used, provided they facilitate peptidepresentation in MHC-class II presentation and T cell activation. Thus,while it is possible for the active ingredients to be administeredalone, they typically are presented as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the presentinvention comprise at least one active ingredient, as above described,together with one or more pharmaceutically acceptable carriers. Aparticular embodiment of the present invention relates to pharmaceuticalcompositions, comprising, as an active ingredient, one or more peptidesaccording to the invention, in admixture with a pharmaceuticallyacceptable carrier. The pharmaceutical composition of the presentinvention should comprise a therapeutically effective amount of theactive ingredient, such as indicated hereinafter in respect to themethod of treatment or prevention. Optionally, the composition furthercomprises other therapeutic ingredients. Suitable other therapeuticingredients, as well as their usual dosage depending on the class towhich they belong, are well known to those skilled in the art and can beselected from other known drugs used to treat immune disorders.

The term “pharmaceutically acceptable carrier” as used herein means anymaterial or substance with which the active ingredient is formulated inorder to facilitate its application or dissemination to the locus to betreated, for instance by dissolving, dispersing or diffusing the saidcomposition, and/or to facilitate its storage, transport or handlingwithout impairing its effectiveness. They include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents (forexample phenol, sorbic acid, chlorobutanol), isotonic agents (such assugars or sodium chloride) and the like. Additional ingredients may beincluded in order to control the duration of action of the monoclonalantibody active ingredient in the composition. The pharmaceuticallyacceptable carrier may be a solid or a liquid or a gas which has beencompressed to form a liquid, i.e. the compositions of this invention cansuitably be used as concentrates, emulsions, solutions, granulates,dusts, sprays, aerosols, suspensions, ointments, creams, tablets,pellets or powders. Suitable pharmaceutical carriers for use in saidpharmaceutical compositions and their formulation are well known tothose skilled in the art, and there is no particular restriction totheir selection within the present invention. They may also includeadditives such as wetting agents, dispersing agents, stickers,adhesives, emulsifying agents, solvents, coatings, antibacterial andantifungal agents (for example phenol, sorbic acid, chlorobutanol),isotonic agents (such as sugars or sodium chloride) and the like,provided the same are consistent with pharmaceutical practice, i.e.carriers and additives which do not create permanent damage to mammals.The pharmaceutical compositions of the present invention may be preparedin any known manner, for instance by homogeneously mixing, coatingand/or grinding the active ingredients, in a one-step or multi-stepsprocedure, with the selected carrier material and, where appropriate,the other additives such as surface-active agents. They may also beprepared by micronisation, for instance in view to obtain them in theform of microspheres usually having a diameter of about 1 to 10 μm,namely for the manufacture of microcapsules for controlled or sustainedrelease of the active ingredients.

Suitable surface-active agents, also known as emulgent or emulsifier, tobe used in the pharmaceutical compositions of the present invention arenon-ionic, cationic and/or anionic materials having good emulsifying,dispersing and/or wetting properties. Suitable anionic surfactantsinclude both water-soluble soaps and water-soluble syntheticsurface-active agents. Suitable soaps are alkaline or alkaline-earthmetal salts, unsubstituted or substituted ammonium salts of higher fattyacids (C₁₀-C₂₂), e.g. the sodium or potassium salts of oleic or stearicacid, or of natural fatty acid mixtures obtainable form coconut oil ortallow oil. Synthetic surfactants include sodium or calcium salts ofpolyacrylic acids; fatty sulphonates and sulphates; sulphonatedbenzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates orsulphates are usually in the form of alkaline or alkaline-earth metalsalts, unsubstituted ammonium salts or ammonium salts substituted withan alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. thesodium or calcium salt of lignosulphonic acid or dodecylsulphonic acidor a mixture of fatty alcohol sulphates obtained from natural fattyacids, alkaline or alkaline-earth metal salts of sulphuric or sulphonicacid esters (such as sodium lauryl sulphate) and sulphonic acids offatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazolederivatives typically contain 8 to 22 carbon atoms. Examples ofalkylarylsulphonates are the sodium, calcium or alcanolamine salts ofdodecylbenzene sulphonic acid or dibutyl-naphtalenesulphonic acid or anaphtalene-sulphonic acid/fomnaldehyde condensation product. Alsosuitable are the corresponding phosphates, e.g. salts of phosphoric acidester and an adduct of p-nonylphenol with ethylene and/or propyleneoxide, or phospholipids. Suitable phospholipids for this purpose are thenatural (originating from animal or plant cells) or syntheticphospholipids of the cephalin or lecithin type such as e.g.phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylglycerine,lysolecithin, cardio-lipin, dioctanylphosphatidylcholine,dipalmitoylphoshatidylcholine and their mixtures.

Suitable non-ionic surfactants include polyethoxylated andpoly-propoxylated derivatives of alkylphenols, fatty alcohols, fattyacids, aliphatic amines or amides containing at least 12 carbon atoms inthe molecule, alkylarenesulphonates and dialkylsulphosuccinates, such aspolyglycol ether derivatives of aliphatic and cycloaliphatic alcohols,saturated and unsaturated fatty acids and alkylphenols, said derivativestypically containing 3 to 10 glycol ether groups and 8 to 20 carbonatoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms inthe alkyl moiety of the alkylphenol. Further suitable non-ionicsurfactants are water-soluble adducts of polyethylene oxide withpoylypropylene glycol, ethylenediaminopolypropylene glycol containing 1to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ethergroups. Such compounds usually contain from 1 to 5 ethyleneglycol unitsper propyleneglycol unit. Representative examples of non-ionicsurfactants are nonylphenol—polyethoxyethanol, castor oil polyglycolicethers, polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethyleneglycol andoctylphenoxypolyethoxyethanol. Fatty acid esters of polyethylenesorbitan (such as polyoxyethylene sorbitan trioleate), glycerol,sorbitan, sucrose and pentaerythritol are also suitable non-ionicsurfactants. Suitable cationic surfactants include quaternary ammoniumsalts, particularly halides, having 4 hydrocarbon radicals optionallysubstituted with halo, phenyl, substituted phenyl or hydroxy; forinstance quaternary ammonium salts containing as N-substituent at leastone C8C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyland the like) and, as further substituents, unsubstituted or halogenatedlower alkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for thispurpose may be found for instance in “McCutcheon's Detergents andEmulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981),“Tensid-Taschenbucw’, 2 d ed. (Hanser Verlag, Vienna, 1981) and“Encyclopaedia of Surfactants, (Chemical Publishing Co., New York,1981). Peptides, homologues or derivatives thereof according to theinvention (and their physiologically acceptable salts or pharmaceuticalcompositions all included in the term “active ingredients”) may beadministered by any route appropriate to the condition to be treated andappropriate for the compounds, here the proteins and fragments to beadministered. Possible routes include regional, systemic, oral (solidform or inhalation), rectal, nasal, topical (including ocular, buccaland sublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intraarterial, intrathecal andepidural). The preferred route of administration may vary with forexample the condition of the recipient or with the diseases to betreated. As described herein, the carrier(s) optimally are “acceptable”in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intraarterial,intrathecal and epidural) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. Such methods includethe step of bringing into association the active ingredient with thecarrier which constitutes one or more accessory ingredients. In generalthe formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as solution or a suspension in an aqueous liquid ora non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with a binder, lubricant, inertdiluent, preservative, surface active or dispersing agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient therein.

For local treatments for example on the skin, such as of the joint, theformulations are optionally applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc),particularly 0.2 to 15% w/w and more particularly 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base. If desired, the aqueous phase of the cream base may include,for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethylsulfoxide andrelated analogues. The oily phase of the emulsions of this invention maybe constituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier (otherwise known as an emulgent), itdesirably comprises a mixture of at least one emulsifier with a fat oran oil or with both a fat and an oil. Optionally, a hydrophilicemulsifier is included together with a lipophilic emulsifier which actsas a stabiliser, typically by including both an oil and a fat. Together,the emulsifier(s) with or without stabiliser(s) make up the so-calledemulsifying wax, and the wax together with the oil and fat make up theso-called emulsifying ointment base which forms the oily dispersed phaseof the cream formulations.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus the cream should optionally be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, and particularly butylstearate, 2-ethylhexyl palmitate or a blend of branched chain estersknown as Crodamol CAP may be used. These may be used alone or incombination depending on the properties required. Alternatively, highmelting point lipids such as white soft paraffin and/or liquid paraffinor other mineral oils can be used. Formulations suitable for topicaladministration to the eye also include eye drops wherein the activeingredient is dissolved or suspended in a suitable carrier, especiallyan aqueous solvent for the active ingredient. The active ingredient isoptionally present in such formulations in a concentration of 0.5 to20%, advantageously 0.5 to 10% particularly about 1.5% w/w. Formulationssuitable for topical administration in the mouth include lozengescomprising the active ingredient in a flavoured basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerine, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier. Formulations for rectal administration may be presented as asuppository with a suitable base comprising for example cocoa butter ora salicylate. Formulations suitable for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns (including particle sizes in arange between 20 and 500 microns in increments of 5 microns such as 30microns, 35 microns, etc), which is administered in the manner in whichsnuff is taken, i.e. by rapid inhalation through the nasal passage froma container of the powder held close up to the nose. Suitableformulations wherein the carrier is a liquid, for administration as forexample a nasal spray or as nasal drops, include aqueous or oilysolutions of the active ingredient. Formulations suitable for aerosoladministration may be prepared according to conventional methods and maybe delivered with other therapeutic agents. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams or spray formulations containing in addition to theactive ingredient such carriers as are known in the art to beappropriate. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Typical unit dosage formulations are those containing a daily dose orunit daily sub-dose, as herein above recited, or an appropriate fractionthereof, of an active ingredient. It should be understood that inaddition to the ingredients particularly mentioned above theformulations of this invention may include other agents conventional inthe art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavouringagents. Peptides, homologues or derivatives thereof according to theinvention can be used to provide controlled release pharmaceuticalformulations containing as active ingredient one or more compounds ofthe invention (“controlled release formulations”) in which the releaseof the active ingredient can be controlled and regulated to allow lessfrequency dosing or to improve the pharmacokinetic or toxicity profileof a given invention compound. Controlled release formulations adaptedfor oral administration in which discrete units comprising one or morecompounds of the invention can be prepared according to conventionalmethods. Additional ingredients may be included in order to control theduration of action of the active ingredient in the composition. Controlrelease compositions may thus be achieved by selecting appropriatepolymer carriers such as for example polyesters, polyamino acids,polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers,methylcellulose, carboxymethylcellulose, protamine sulfate and the like.The rate of drug release and duration of action may also be controlledby incorporating the active ingredient into particles, e.g.microcapsules, of a polymeric substance such as hydrogels, polylacticacid, hydroxymethylcellulose, polymethyl methacrylate and the otherabove-described polymers. Such methods include colloid drug deliverysystems like liposomes, microspheres, microemulsions, nanoparticles,nanocapsules and so on. Depending on the route of administration, thepharmaceutical composition may require protective coatings.Pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation thereof. Typical carriers for this purpose therefore includebiocompatible aqueous buffers, ethanol, glycerol, propylene glycol,polyethylene glycol and the like and mixtures thereof. In view of thefact that, when several active ingredients are used in combination, theydo not necessarily bring out their joint therapeutic effect directly atthe same time in the mammal to be treated, the corresponding compositionmay also be in the form of a medical kit or package containing the twoingredients in separate but adjacent repositories or compartments. Inthe latter context, each active ingredient may therefore be formulatedin a way suitable for an administration route different from that of theother ingredient, e.g. one of them may be in the form of an oral orparenteral formulation whereas the other is in the form of an ampoulefor intravenous injection or an aerosol.

The experimental part of the present invention shows a subset ofantigen-specific, in vivo induced and easy to expand T cells withregulatory properties. The latter include: (1) induction of APCapoptosis after MHC-class II dependent cognate activation, affectingboth dendritic and B cells, and demonstrated in vitro and in vivo, and;(2) suppression of bystander T cells by a contact-dependent mechanism inthe absence of IL-10 and/or TGF-13. The present invention furtherdiscloses methods to distinguish induced cytolytic Tregs from bothnatural and adaptive Tregs. Based on characterisation of 15 clones, thesurface phenotype is defined as CD25hi, CTLA-4-hi, GITR+ and ICOS+, butCD127(−). Such clones express low levels of CD62L and CD103 but notCCR7. Cytokine production was limited to IFN-gamma, with no TGF-beta orIL-10. All clones were Foxp3(−) but strongly positive for T-bet andEgr-2, together with high transcript levels for granzyme B. NaturalTregs are defined by expression of Foxp3 and absence of CD127, producehigh levels of IL-10, at least in vitro, and are specific forautoantigens. Among the numerous adaptive T cells described so far, mostshare the production of IL-10 (and TGF-beta in some cases such as in Th3cells) and are Foxp3 and CD25 negative. Central to cytolytic Tregs isthe strong expression of T-bet. T-bet is induced by IFN-γ via aSTAT1-dependent pathway and exerts different activities, includingsuppression of IL-2 transcription, induction of granzyme transcriptionand is a maturation factor for Th1 differentiation.

No transcript for IL-2 was found in the T cell clones of the presentinvention and, interestingly, exogenous IL-2 did not restore IL-2transcription, even after repeated stimulation cycles in vitro. Thissuggests that cytolytic Tregs have undergone an epigenetic alteration,which would sustain their regulatory activity both in vitro and in vivo.Although T-bet is required for the maturation of the Th1 subset, itsexpression does not necessarily qualify cells as belonging to suchlineage. Induction of granzyme B by T-bet has been observed with CD8+cytolytic T cells. The Tregs described here are anergic in the sensethat they were unable to proliferate and/or produce IL-2 uponcross-linking of their antigen receptor. Interestingly, this anergicstate could be overcome by addition of IL-2. A high expression wasobserved of the transcription factor Egr-2 (Krox-20), which upregulatescell cycle inhibitors such as p21^(cipl) and p27^(kip). It is known thatIL-2 can override Egr-2-mediated suppression, possibly via activation ofNF-kB. The latter upregulates granzyme transcription, which mightsynergise with T-bet for the production of granzyme. It should be keptin mind that the regulatory properties are not lost when cells arestimulated by addition of IL-2. In a particular embodiment, this is aproperty of the present cytolytic Tregs, a seemingly stable cell subsetthat exerts its regulatory activity upon IL-2 dependent activation.Induction of apoptosis is the mechanism at the basis of regulatoryactivity. This was demonstrated at both the level of APC and bystander Tcells by showing activation of caspase 3 and/or annexin V binding. Itwas shown that the two main pathways leading to apoptosis were activatedin cytolytic Tregs. Both GrB and perforin production were induced byactivation of Tregs, with increased transcription for GrB but notgranzyme A. GrB, as opposed to GrA, induces apoptosis by at least twomechanisms, namely direct and indirect activation of pro-caspase 3, thelatter through the release of cytochrome C by mitochondria and caspase 9activation. Specific inhibitors of GrB showed significant reduction inapoptosis induction, and only at concentrations close to cellcytotoxicity. Whether perforin is required for such an activity isdoubtful, as EGTA did not block apoptosis to any significant degree. Thesecond pathway by which apoptosis can be induced is the Fas-FasLpathway. Interestingly, FasL is present in exocytose granules togetherwith granzymes, and anchors to the membrane upon cell activation, whichconstitutes the main pathway for FasL expression in T cells. FasLsignals through Fas leading to caspase 8 activation, with a downstreamactivation of caspase 3 and caspase 9 through mitochondrial release ofcytochrome C, thereby synergising with GrB. Partial inhibition ofapoptosis was obtained using a FasL-specific antibody. The relativeparticipation of the GrB and FasL pathways is dictated by the extent ofFasL expression. Thus, Wehi cells, which have a constitutive highexpression of Fas are readily lysed by Tregs, as compared for instancewith dendritic cells. Whether the combined action of GrB and FasLaccounts for all cytolytic activity is not fully established, aspreliminary experiments have shown that the combination of the twoinhibitors did not abolish target cell apoptosis. Granzymes are known tobe secreted by some Tregs. Thus, granzyme B is involved in the mechanismby which natural Tregs control immune responses, both in human and inthe mouse. GrB KO mice have a defect in regulation. It is, however,difficult to establish to what extend induced Tregs use granzymes toexert their regulatory activity. One report shows that a proportion ofTr1-like Tregs activated by anti-CD3 and anti-CD46 antibodies expressGrB. A yet-to-be solved difficulty with granzymes is the lack ofspecific and efficient inhibitors. Chemical or peptide inhibitors, suchas those used in the present invention (Example 11), required highconcentrations to be active. The use of the Fas-FasL pathway has notbeen earlier reported in adaptive Tregs, and very few data indicate thatnatural CD4+CD25+ Tregs could also use Fas-L as mechanism. Noteworthy,apoptosis induction is observed with dendritic cells and with B cells,suggesting that both primary and secondary immune responses can beregulated. In addition, Tregs recognising a single T cell epitope fromeven complex antigens have the capacity to suppress the response to theentire protein by eliminating the antigen-presenting cell. This is wellillustrated by the in vivo data, in which the response toward a fullallergen, Der p 2, is suppressed after adoptive transfer of a singleTreg clone. This effect is reinforced by the suppression of bystander Tcells even when the latter are activated by interaction with a differentAPC, provided cell contact is possible between Tregs and effector Tcells. Interestingly, Tregs can regulate effector T cells at variousmaturation stages, Th0, Th1 or Th2. Importantly, cytolytic Tregs induceapoptosis in target cells and not necrosis. Apoptotic APC may play arole in suppression. It has indeed been demonstrated that apoptoticcells taken up by antigen-presenting cells induce tolerance, whilenecrotic cells rather induce inflammation. In vivo, the nearly completesuppression of inflammation within lungs should certainly be consideredas a token for target cell apoptosis instead of necrosis. An aspect ofthe present invention is the demonstration that B cell apoptosis alsooccurs in vivo. Thus, mice adoptively transferred with transgenic Bcells expressing p21-35 followed by cytolytic Tregs of correspondingspecificity show complete disappearance of B cells, as detected in thespleen. It is unlikely that transgenic B cells would have migrated toother sites. No evidence for such cells was found in lungs or in theliver. The functional properties of p21-35 transgenic cells areidentical to those of B cells incubated with the peptide in aconventional loading assay. It is shown here that transgenic B cells areinduced into apoptosis in vitro by co-culture with cytolytic Tregs,indicating good evidence for the in vivo relevance of APC cytolysis.Particular attention was paid to the possible involvement of IL-10.Natural Tregs as well as most if not all described subsets of adaptiveTregs produce IL-10 (Levings et al. (2002) Int. Arch. Allergy Immunol.129, 263-276. One of these subtypes was induced after respiratoryexposure to allergen and expressed Foxp3, GATA3, and produced no IFN-γ(Akbari et al. (2002) Nature medicine 8, 1024-1032). Another type wasinduced during strong Th1 polarising conditions (Listeria monocytogenesas adjuvant), expressed Foxp3, T-bet, and produced IFN-γ (Stock et al.(2004) Nat. Immunol. 5, 1149-1156). Both subsets were able to inhibitairway hyper-reactivity and inflammation via an IL-10 dependentmechanism. There was no evidence for IL-10 production by the Foxp3negative cytolytic Tregs nor activation of STAT3 or SP1. The observationthat the induction of apoptosis required direct cell contact as shown intranswell experiments, and the known suppression of IL-10 transcriptionby IFN-gamma, the latter being produced at high levels by all of thecytolytic Tregs, were taken as evidence against a significantinvolvement of IL-10 in cytolytic Treg activity. Remarkably, a mildadjuvant such as alum is all that was required. Interestingly,immunisation with the mp 21-35 peptide made in CFA/IFA was even moreeffective in suppressing airway inflammation and hyperreactivity, butthis was to the detriment of accumulation of large numbers of Th1lymphocytes into lungs. In addition, this shows that immunisation in CFAfailed to elicit cytolytic Tregs. Methylation of cysteine increased MHCclass II presentation, but this is attributable to increased stabilityof peptide and increased uptake by APC. Interestingly, it was notpossible to induce cytolytic Tregs towards a second major T cell epitopeof Der p 2, p71-85. Substitution of isoleucine 28 by asparagine reducedbinding affinity to MHC class II molecules, which was shown not to bedetrimental to the elicitation of cytolytic Tregs. Altered peptidescarrying T cell epitopes have been reported to either increase bindingaffinity and/or TCR recognition, or to induce tolerance, an outcomeoften difficult to predict. In the present case, both effector andcytolytic Tregs recognised the same epitope, and could be expanded invitro with p21-35 in either its wild type or mutated sequence.

An interesting aspect of the present study was the demonstration thatcytolytic Tregs migrated towards the lung upon airway exposure to theallergen Der p 2, and this, in the absence of peripheral allergensensitisation. Chemokines involved in attracting T lymphocytes to thelung are not precisely identified. The cytolytic Tregs described hereexpress CD103 but not CCR7, which should confer them with the capacityto migrate to inflamed tissues. Other chemokines such as CCR5 and CCR3were not detected. However, the model system used here, in which miceadoptively transferred with cytolytic Tregs are submitted to 2 series of3 nasal instillations at one-week interval, does not elicit significantinflammation in the lungs, as shown in control mice submitted toallergen inhalation only. In an experimental model of asthma, includingperipheral sensitisation and allergen inhalation, it was shown thatTregs have the capacity to prevent and suppress both inflammatoryinfiltration and airway hyperreactivity, which are the hallmarks ofbronchial asthma. An LPS-free recombinant form of a main allergen, Der p2, was used, which is involved in a large proportion of patientssuffering from allergic asthma. In parallel studies, it was determinedthat the p21-35 peptide activated CD4+ effector T cells from patientssensitised to Der p 2. A p21-35 specific T cell clone derived from sucha patient shows properties comparable to those of mouse cytolytic Tregs,which suggests that cytolytic Tregs are generated as part of a normalimmune response to an allergen such as Der p 2.

These observations support the utility of cytolytic Tregs according tothe invention for treating allergic asthma in the clinic. CytolyticTregs are activated and easily expanded by MHC class II-restrictedepitope presentation, and do not produce suppressive cytokines, whichprovides the specificity required for clinical use. In addition, suchTregs are obtained upon immunisation using conventional adjuvant, namelyalum, with no requirement for bacteria-derived material such asmycobacterium or CFA. Most encouraging is the observation that airwayhyperreactivity to a non-specific stimulant, namely metacholine, wassignificantly reduced even after allergen sensitisation. This wasunexpected, as airway inflammation and hyperreactivity are notnecessarily linked. In fact, in the model of asthma to Der p 2, the twophenomena are dissociated from each other.

The present invention will now be illustrated by means of the followingexamples which are provided without any limiting intention. Furthermore,all references described herein are explicitly included herein byreference.

EXAMPLES Example 1 P21-35 Contains a Thioredoxin/Glutaredoxin ConsensusSequence

Table 1 shows the amino acid sequence homology between a fragment ofp21-35 and C—X(2)-S redox motifs in proteins from different organisms.

FIG. 1 shows the capacity of p21-35 to reduce disulfide bridges in theinsulin reduction assay (turbidimetric assay). Herein, an insulinsolution (1 mg/ml) containing DL-Dithiothreitol was incubated with areducing protein or peptide or control for 20 minutes at 25° C. Theincrease in optical density at 650 nm (Y axis) due to precipitation ofreduced insulin at pH 7 was then measured at different time points (Xaxis). A recombinant tetramer of p21-35 (B4) [SEQ ID. NO: 4] was usedwith recombinant thioredoxin as positive control.

Example 2 Uptake of p21-35 by Antigen-Presenting Cells is Increased byAddition of a Subdominant T Cell Epitope (FIG. 2)

Induction of apoptosis of WEHI B cells was used as a measure of antigenuptake and presentation. WEHI B cells (2×10⁴) where mixed with a peptidep21-35 specific Treg clone (ratio 1/1) and decreasing concentrations ofpeptide p21-35 (X axis), or the same peptide linked to a minor T epitopefrom tetanus toxin (p830-844; T-B). After 18 hours, cells were labelledwith anti-CD19-PE antibodies (a marker of WEHI cells) and AnnexinV-FITC, an apoptosis marker. Samples were then analysed by flowcytometry. Results are representative of the proportion of WEHI cellspositive for Annexin V. A 100-fold increase in the capacity to induceapoptosis was observed when peptide p21-35 was linked to a minor T cellepitope, as indicated in FIG. 2.

Example 3 Amino Acid Residues Involved in the Thioredoxin-Like ConsensusSequence are Instrumental for Expression of Regulatory Properties

Panel A of FIG. 3 shows the location of the thioredoxin-like sequencemotif within peptide p21-35. The CHGS [SEQ ID. NO: 3] sequence isadjacent to the MHC-class II binding cleft. Alanine substitution ofresidue 21C or residue S24 of the thioredoxin-like C—X(2)-S motif doesnot impair T cell epitope recognition as measured by the proliferation(3H-thymidine incorporation) of a Treg clone, is indicated in panel B ofFIG. 3. Proliferation was carried out using 5×10⁴ WEHI cells incubatedfor 1 h at 37° C. with 6 μM of peptide. The cells were then washed andco-cultured for 4 days with 5×10⁴ T cells. ³H-thymidine was added 24 hbefore the end of the culture. Radioactivity was counted on cellsadsorbed on fibre glass filters. Substitution of residues 21 and 24abrogate the capacity of the Treg clone to lyse antigen-presenting cellsin an apoptosis induction assay (see panel C of FIG. 3) carried out asdescribed in Example 2.

Example 4 Generation of Tregs with Physiological Properties UponInjection of T-Bb in Mice

The capacity of the peptide T-B to prevent Der p 2-induced asthma wasevaluated as follows. A group of 8 BALB/c mice (indicated as Tbalum inFIG. 4) received 3 footpath injections (20 μg per injection) carried outat 2-week interval with the T-B peptide adsorbed on alum. Two weeksafter the last injection, all mice received IP injections ofalum-adsorbed full length rDer p 2 (40 μg per injection) on 3 occasionswith a 2-week interval, followed by nasal instillations of rDer p 2 insaline (100 μg rDer p 2 in 50 μl PBS per instillation). The results wereevaluated by comparison with a group of mice treated by rDer p 2(indicated as “Der p 2 model” in FIG. 4) but which did not receivepeptide injections. The Figure indicates that pre-immunisation with thepeptide significantly reduces BAL cell counts evaluated on cytospins(FIG. 4 panel A), lung histology scores (FIG. 4, panel B) and airwayhyperreactivity (FIG. 4 panel C) assessed by reactivity to increasedconcentrations of methacholine.

Example 5 Effect of P21-35 on the Oxidative Metabolism of Cognate TregCells

A Treg clone (10^(5 cells)) specific for p21-35 was incubated for 90minutes with PBS (FIG. 5, panel A), peptide p21-35 (20 μg/ml in 200 μlPBS; FIG. 5, panel B) or tert-butyl hydroperoxide (100 μM in PBS; FIG.5, panel C), which is an inducer of Reactive Oxygen Species (ROS). Cellswere then labelled for 30 minutes with carboxy-H2DCFDA (12 μM), afluorogenic marker for ROS in live cells, and then analysed by flowcytometry. The picture shows that peptide p21-35 stimulates theoxidative metabolism of cognate Treg cells and induces a doubling in thefluorescence intensity due to ROS increase.

Example 6 TREG Cell Clones Exhibit Cytotoxic Properties onAntigen-Presenting Cells

The cytotoxic properties of a Treg line (G121) was tested on the WEHI Bcell line used as an antigen-presenting cell. FIG. 6 shows the % ofcells lysed over a 14-h period of time in the presence of the cytotoxiccell line alone (WEHI (10⁴ cells)+T cell (10^(4 cells))), the T cellline with addition of the p21-35 peptide (WEHI+T cell+p21), or WEHIcells pre-loaded with the p21-35 peptide (WEHIp21+T cell). Lysis of WEHIcells was measured using the JAM assay (a quantitative assay of DNAfragmentation): 10⁴ WEHI cells were pre-incubated with ³H-thymidine (4.5pCu/ml, 1 ml) for 10 h at 37° C. and then washed before co-culture withthe Treg line. The release of ³H-thymidine in supernatants was taken asa measure of WEHI cell lysis. The data shows that the presence of thepeptide is required to activate the T cell line.

Example 7 TREG Cell Clones Suppress the Activation of T Cells Specificfor Another Epitope on the Same Antigen, or Specific for Another Antigenby Cytotoxicity (FIG. 7)

T cell lines (TCL; 10⁵ cells) specific for Der p 1, an allergenunrelated to Der p 2 or specific for an alternative major T cell epitope(p71-85) located on Der p 2 were labelled with CFSE (which is a labelfor cytoplasmic proteins, making it possible to evaluate the number ofcell divisions based on intensity of staining, which reduces to 50% eachtime the cell divides) (12.5 nanoM) (FIG. 7). The proliferation of suchCFSE-labelled Der p 1 specific TCL (Panel A and B) or p71-85 TCL (panelC and D) was measured before (panel A and C) or after (panel B and D)incubation with the cytotoxic Treg clone (ratio 1/1). Antigen-presentingcells were preloaded with both the specific allergen (Der p 1 or Der p2; 0.5 ml containing 10 μg/ml in each case) and peptide 21-35 (0.5 mlcontaining 10 μg/ml). After 72 hours of incubation, cells wereharvested, stained with propidium iodide and analysed in a flowcytometer. Histograms and percentages in parts B and D of the Figurerepresent the proportion of residual living CFSE-positive cells (livecells were discriminated by being propidium iodide negative). The Figureshows that the cytotoxic Treg clone specific for peptide 21-35 stronglydecreased the proliferation of each of the 2 bystander T cells (CFSEdivisions). Most of the CFSE-stained cells became positive for theapoptosis marker propidium iodide, indicating that bystander T cellswere killed in the assay.

Example 8 Determination of the Phenotypic Profile of Treg Cells

FIG. 8 shows cytokine production of four p21-35 specific Treg clonesderived from mice treated with the peptide T-B composition as in Example4 (left panel). Supernatants of cell culture were analysed for cytokinecontent after four days of stimulation with antigen-presenting cells(irradiated splenocytes from naïve mice, 10⁵ cells) and peptide p21-35(2 μg/ml, 200 μl). Treg clones mainly produced IFN-γ (IFN-G) and onlytrace amounts of TNF-α (TNF-a) and IL-10. The right panel shows them-RNA analysis of such Treg cells. Transcripts for transcriptionrepressor Foxp3 were not detected, but T-bet, Granzyme A and Granzyme Bshow strong transcription levels.

FIG. 9 shows the expression levels of various genes in a 21-35 peptidespecific T cell clone at rest by fluorescence-activated cell sorting(Facs). In Table 4, the mean fluorescent intensity is provided of fourdifferent clones (T1 to T4) (2×10⁵ cells). High levels of CD25, ICOS,GITR, CD103 and intracellular CTLA-4 were observed. CD28 was not orpoorly expressed. CD62-L and CD45RB were expressed at low levels. Allantibodies were from Becton-Dickinson (NJ, USA).

TABLE 4 mean fluorescence of fluorescent antibody labelled Treg clonesCD28 CD62L CD103 CD45RB ICOS GITR CTLA-4 T1 5 36 21 6 251 127 98 T2 1227 25 10 245 150 110 T3 4 39 27 6 263 110 92 T4 4 36 14 7 253 129 87

Example 9 Treg Cells with Cytotoxic Properties are Elicited byImmunisation with a Peptide Made of a Dominant T Cell Epitope of theAllergen Der P 1 Linked to a Glutaredoxin-Like Consensus Sequence (FIG.10)

A dominant T cell epitope of the allergen Der p 1 recognised by BALB/cmice SNYCQIYPPNANKIR p114-128 [SEQ ID. NO: 5] was synthesised in linewith the sequence CGFS (motif sequence [SEQ ID. NO: 6]), which carries aglutaredoxin-like consensus sequence from E. coli k12. The latter wasadded at the amino-terminal end of the T cell epitope (taking s114 asP1).

The full peptide has the sequence CGFSSNYCQIYPPNANKIR [SEQ ID. NO: 7].The sequence containing the Der p 1 T cell epitope without theglutaredoxin-like consensus sequence [SEQ ID. NO: 5] was used as acontrol.

Both peptides were joined by a linker sequence SGGSGGSGG [SEQ ID. NO: 8]at the amino-terminal end of the sequence to the amino terminal end ofthe sequence IITIAWAALLLVAAIFGVASCLI RSRSTKNEANQPLLTDS [SEQ ID. NO: 9],corresponding to the transmembrane domain and the truncated cytosolictail of the gp75 protein, in order to target the T cell epitope to thelate endosome (the dileucine-based motif is underlined).

The full sequence of the peptide comprising targeting sequence, linker,motif and epitope sequence is:

CGFS SNYCQIYPPNANKIR SGGSGGSGG

IITIAWAALLLVAAIFGVASCLIRSRSTKNEANQPLLTDS [SEQ ID. NO: 10], whereas thecontrol peptide without the sequence CGFS [SEQ ID. NO: 6] has thesequence:

[SEQ ID. NO: 11] SNYCQIYPPNANKIR SGGSGGSGGIITIAVVAALLLVAAIFGVASCLIRSRSTKNEANQPLLTDS

Groups of BALB/c mice were immunised subcutaneously (20 μg) with theexperimental peptide [SEQ ID. NO: 10] or the control peptide [SEQ ID.NO: 11] adsorbed onto aluminium hydroxide. Three injections wereperformed at 2-week intervals. Ten days after the last immunisation,mice were sacrificed and CD4+ T cells prepared from the spleen usingmagnetic beads. CD4+ T cells (2×10⁶ cells) were then stimulated in vitroby the Der p 1 T cell epitope (20 μg) presented by adherent spleen cellsserving as antigen-presenting cells.

Ten days after stimulation the number of specific T cell lines obtainedin each group was calculated by limiting dilution analysis. Each cellline was evaluated for its capacity to lyse WEHI cells, a B cell lineselected for its efficacy in antigen-presentation by MHC class-IIdeterminants, as described in Example 6. Only cells obtained fromanimals immunised with the peptide containing the glutaredoxin consensussequence had acquired the capacity to lyse WEHI cells, and lysis occursonly in the presence of the cognate Der p 1 T cell epitope.

FIG. 10 shows that T cell clones with Treg properties could only bederived from mice that received the construct made of the Der p 1epitope linked to a glutaredoxin-like redox motif and gp75, but not frommice that received the control peptide. All T cell clones obtained aftertreatment with the construct and re-stimulated in vitro were cytotoxic.

Example 10 Use of a Dominant T Cell Epitope of MOG Protein in an In VivoModel for Multiple Sclerosis

Multiple sclerosis can be induced in experimental models by immunisationwith the Myelin Oligodendrocyte Glycoprotein (MOG) peptideVGWYRSPFSRVVHLYR [SEQ ID. NO: 12], which corresponds to amino acidresidues 37-52 of the MOG protein. This peptide contains a dominant Tcell epitope. The P1 position, i.e. the first amino acid anchored intothe MHC class II groove is Y40 (the P1-P9 sequence is underlined). The 3amino acids of the amino terminal end of the peptide are replaced by thesequence CGPS [SEQ ID. NO: 13], which corresponds to the humanthioredoxin sequence, residues 21 to 24), resulting in the peptideCGPSYRSPFSRVVHLYR [SEQ ID. NO: 14]. The experimental peptide [SEQ ID.NO: 14] and the control peptide [SEQ ID. NO: 12] are joined by thelinker sequence SGGSGGSGG [SEQ ID. NO: 8] at the amino-terminal end ofthe sequence VSVSAVTLGLGLIIFSLGVIS WRRAGHSSYTPLPGSNYSEGWHIS [SEQ ID. NO:15] corresponding to the transmembrane domain and the cytosolic tail ofthe HLA-DMβ protein, in order to target the T cell epitope to the lateendosome (the tyrosine-based peptide motif is underlined).

The sequences of the targeted peptide are:

[SEQ ID. NO: 16] VSVSAVTLGLGLIIFSLGVISWRRAGHSSYTPLPGSNYSEGWHIS SGGSGGSGGCGPS YRSPFSRVVHLYR and [SEQ ID. NO: 17]VSVSAVTLGLGLIIFSLGVISWRRAGHSSYTPLPGSNYSEGWHIS SGGSGGSGG YRSPFSRVVHLYR

A group of C57BL/6 mice is adoptively transferred with a CD4+MOG-specific effector T cell clone following a protocol meant to inducea multiple sclerosis-like syndrome. This involves administration of theMOG peptide in complete Freund's adjuvant and 2 injections of Pertussistoxin. This protocol elicits an expansion of the effector T cell clone,which results in the development of signs compatible with multiplesclerosis within 12 days after the MOG peptide administration.

A second group of C57BL/6 mice is first adoptively transferred with aMOG-specific regulatory T cell clone (obtained using the peptides [SEQID. NO: 16 and 17] described above), followed after 1 day by the fullprotocol of disease induction.

It is observed that the clinical score developed by mice pre-treatedwith a cytolytic T cell clone is significantly reduced as compared tomice receiving only the effector T cell clone.

Methods and Materials

Mice. Six to eight-week-old BALB/c mice were obtained from in-housefacilities. The in vivo studies were approved by the University ofLeuven's Ethical Committee.

Reagents. Peptides derived from Dermatophagoides pteronyssinus group 2allergen (Der p 2) Der p 1 and tetanus toxoid were synthesised(purity, >85%). Sequences are: p21-35, CHGSEPCIIHRGKPF [SEQ ID. NO: 2];p114-128 (amino-acids 114-128 from Der p 1), SNYCQIYPPNANKIR [SEQ ID.NO: 5]; p830 (amino-acids 830-844 from tetanus toxoid), QYIKANSKFIGITEL[SEQ ID. NO: 18]; mp 21-35 QYIKANSKFIGITELGGCHGSEPCIIHRGKPF [SEQ ID. NO:19]; mp 21-35Asn QYIKANSKFIGITELGGCHGSEPCNIHRGKPF [SEQ ID. NO: 20].Recombinant full-length Der p 2 was produced in Pichia pastoris.

Allergen sensitisation. Animals were sensitised on day 1, 14 and 28 byan i.p. injection of 40 μg recombinant allergen absorbed on 6 mgAL(OH)₃. On day 43, 44, and 45, mice were exposed to 100 μg allergen in50 μl saline by intranasal instillation, followed by a first measurementof pulmonary function by whole body plethysmograph. A second series ofnasal instillations was performed on day 50, 51 and 52 followed byassessment of pulmonary function by whole body plethysmograph.

Cell purification. Splenic CD4 T cells were obtained after peptideimmunisation. After Ficoll density gradient purification (NycomedPharma), they were enriched by negative selection using the CD4 T cellIsolation Kit (Miltenyi Biotec) and LS separation columns. Splenicdentritic cells were positively selected using CD11c Microbeads(Miltenyi Biotec) on LS separation columns. They were stimulated with 5μg/ml LPS (Sigma) for 5 hours, then washed and kept for 18 hours in aCO2 incubator; live cells were recovered by eliminating dead andapoptotic cells with annexin V microbeads on LS columns and MidiMacs(all from Miltenyi Biotec).

B lymphocytes were prepared from spleen cells of naive BALB/c mice bypositive selection using CD19 microbeads (Miltenyi Biotec). As antigenpresenting cells, splenocytes were depleted from T cells by CD90microbeads (Miltenyi Biotec) on LD depletion columns. In some assays,splenic adherent cells were prepared by incubating splenocytes for 2 hat 37° C. in culture medium. Non-adherent cells were removed and theremaining cells were recovered for assays.

Lung lymphocytes were prepared by collagenase (Sigma) digestion andPercoll (Pharmacia) centrifugation as previously described (Abraham etal. (1990) J. Immunol. 144, 2117-2122).

Cell culture. Dendritic cells, T cells and B cells were cultured in RPM'1640 medium containing 5% FCS, 50 μM 2-ME, 200 μg/ml Gentamicin(Invitrogen). Wehi 231 cells were purchased from the European collectionof cell cultures (ECACC).

The effector T cell clone G221N is specific for peptide p21-35. G121,R3TB7, T1 and T3 identify cytolitic T cell clones tested in this study(in Example 11).

Peptide processing assays. G221N, an effector T cell clone specific forpeptide p21-35 was tested in a proliferation assay were splenic adherentcells were used as APC. They were pretreated with either 0.2 μMleupeptin, 0.1 mM chloroquine, 60 μM colchicin or left untreated for 30min. After 3 washes with PBS, APC were loaded for 1 hour with peptidep21-35, mp 21-35 or Der p 2 at 37° C. They were then washed twice withculture medium and added to the G221N clone (10⁵ each) for 72 h. Toblock endocytosis APC were also treated with NaN3/deoxyglucose (2 mg/ml,50 mM, respectively) during all the incubation time with the peptidesfollowed by 3 washes with cold PBS.

Proliferation was assayed by the addition of 1 μCi/well of [³H]thymidine(ICN) during the last 18 h. Cells were harvested and incorporatedisotope counted (cpm). Data were expressed as stimulation indexcalculated by dividing the cpm obtained for G221N T cells stimulatedwith peptide loaded APC by the value obtained with unloaded APC.

Derivatisation of regulatory T cell clones. BALB/c mice were immunisedby 3 footpad injections of 20 μg/ml peptide mp 21-35Asn in alum at 2weeks intervals. Ten days after the last injection, spleen CD4+ T cellswere stimulated with T cell depleted splenocytes from naïve mice in thepresence of peptide mp 21-35Asn. After 10 days, cells were restimulatedin the same conditions but with 10 U/ml mouse 11-2 (Roche). After thefifth restimulation, T cells were subcloned in the presence of 10 U/mlIL-2 by limiting dilution. The subsequent specific stimulations werecarried out in the presence of 20 μml mouse IL-2. The G121 T cell linewas derived as previously described (Janssens et al. (2003) J. Immunol.171, 4604-4612.

FACS analysis. A FACSCalibur (Becton Dickinson) was used for analyticalflow cytometry and data were analysed with CellQuest software. Ten daysafter the last restimulation, T cells were stained with antibodies toCD25, CD28, CD62-L, CD103, CD45RB, ICOS, CTLA-4, and CD11c,(Pharmingen), GITR, Foxp3, Granzyme-B, T-bet(4B10), perforin, CD127, andVb8.1 TCR, (eBioscience).

Bystander suppression assays. Target CD4+CD25− T cells and T helperclones were labelled with 125 nM CFSE (Molecular Probes) for 15 minutesin PBS at 37° C. The reaction was stopped by washing the cells with PBScontaining 30% FBS. These cells (3×10⁵) were cocultured with 10⁵cytotoxic CD4+ T cell clones and T cell depleted splenocytes with 1μg/ml anti-CD3 antibody (eBioscience). For suppression of T helperclones, 10⁵ cells were co-cultured with the same number of cytotoxiccells and T cell depleted splenocytes. After 48 h or 72 hours, cellswere collected and analysed by flow cytometry.

For some cultures, blocking antibodies against FAS-L, GITR, LAG-3 wereused at 10 μg/ml (eBioscience). Transwell assays were performed in 24well plates (Becton Dickinson).

Apoptosis detection. Annexin V-FITC or -PE were used to detect celldeath in B cells, dendritic cells and T cells (Annexin V detection kit,BD Biosciences). In some experiments, apoptosis was measured byintracellular detection of activated caspase-3 with FITC- or PE-labelledantibodies (Pharmingen) or by nuclear labelling with a propidium iodide(PI) staining solution (Pharmingen) according to manufacturerinstructions.

For inhibition of GZ-B activity, Z-AAD-CMK (Calbiochem) or3,4-dichloroiso-coumarin (DCIC) (SIGMA) were added at indicatedconcentrations during all the co-culture period.

Cytokine detection. One million Treg cells were restimulated with 3million irradiated T cell-depleted splenocytes for 72 hours.Supernatants were assessed for the presence of different cytokines.IL-10 and IL-13 were evaluated using the OptElA mouse Elisa kit (BDBiosciences). TGF-β and IL-13 were evaluated with the DuoSet anti-mouseTGF-b1 or DuoSet anti-mouse IL-13 assay kits, respectively (R&DSystems). II-2, IL-4, IL-5, IFN-γ and TNF-α production were analysed byflow cytometry using the Th1/Th2 Cytokine CBA kit (BD Biosciences).

For polyclonal CD4+ cells stimulation, 10⁶ cells were stimulated with5×10⁵ irradiated T cell depleted splenocytes (from naïve mice) and 10μg/ml Der p 2 for 72 h.

Polyclonal CD4+ cell proliferation. 10⁵ Splenic CD4 cells from peptidetreated mice were stimulated with 10⁵ T cell depleted splenocytes and 10μg/ml mp 21-35, 5 μg/ml peptide p830 or 10 μg/ml Der p 2. (3H)-thymidineincorporation was assayed as already described. Results are shown asaverage isotope counting (cpm)±s.e.m. from 6 mice individually tested intriplicates.

Airway hyper-reactivity. Airway hyper-reactivity (AHR) was measured inunrestrained mice using a whole body plethysmograph (EMKA) according topublished methods (Hamelmann et al. (1997) Am. J. Resp. Crit. Care Med.156, 766-775). The peak enhanced pause (PenH) was used as a parameterfor bronchoconstriction. Animals were exposed for 1 minute to increasingdoses of aerosolised methacholine (from 10 to 100 mg/ml), followed by 3minutes rest during which breathing parameters were evaluated. PenHvalues were expressed as means of measurements carried out every 30seconds over a 3-minute period after each methacholine exposure. Lungcompliance were measured with FlexiVent system (Scireq).

Bronchioalveolar lavage fluid collection (BALF). Three days aftermethacholine challenge, mice were sacrificed, the trachea was isolatedand a canula was inserted. BALF was collected by washing with 1 ml ofsaline containing 5% BSA (used for cytokine detection) and then followedby 2×1 ml of saline. Cell count was established. Cytospins were preparedby centrifugation at 1400 rpm for 6 min and stained (Diff-Quik method).One hundred cells were counted in 3 different fields for cellidentification.

Lung Histology. Lungs were fixed with 4% formaldhyde, dehydrated andembedded in paraffin for sectioning (7 μm-thick slides) and stained withhematoxylin/eosin. Eosinophils were detected by May-Grünwald Giemsastaining. Goblet cells in airway mucosa were identified by the periodicacid-Schiff reaction (PAS). PAS positive cells were counted andexpressed as percentage of total epithelial cells. For each mouse, 5fields were examined from each lung section, from the central bronchi aswell from small bronchi.

The density of eosinophils and lymphocytes infiltration was graded asfollow: absent: 0; light but not systematic: 1; light: 2; light tomedium: 3; medium: 4; medium to high: 5; high: 6.

All slides were examined by two persons, including a pathologist who wasunaware of the groups to which mice belonged.

For analysis of transferred T cells, lungs were fixed withparaformaldehyde, cryoprotected with 20% sucrose overnight and snapfrozen in OCT media. Cryostat sections (9 μm) were cut, fixed in acetoneand mounted with antifade reagent (ProLong Gold; Invitrogen) andanalysed with confocal microscopy. Analysis was performed on a ZeissAxioplan2 connected to a 3CCD video camera (DXC-930P, Sony), and KS300software (Zeiss).

Targetted expression of peptide in B lymphocytes. The onco-retroviralpMND-SN vector was obtained from Dr. D. Kohn, USC. A fusion constructcontaining p21-35 (amino acids 21 to 35 of Der p 2) and thecarboxy-terminal end of gp75 (amino acids 488 to 539) connected with alinker, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly [SEQ ID. NO: 8], was made byPCR and cloned into pMND vector. The resulting vector pMND-p21gp75 wasused in conjuction with vectors coding for viral proteins to transientlytransfect 293T cells and onco-retroviral producer cells were obtained aspreviously described (Janssens W. et al., Human gene therapy 14, 263-276(2003)). Splenic B cells preactivated for 24 hours with 50 μg/mlbacterial LPS were cocultured with filtered viral vector-containingsupernatant in the presence of polybrene (6 μg/ml) and LPS (50 μg/ml). Bcells were then washed extensively before adoptive transfer to naïveBALB/c mice.

Analysis of mRNA. Polymerase chain reaction (PCR) and reversetranscriptase PCR (RT-PCR) were performed as previously described(Janssens W. et al. (2003), Human gene therapy 14, 263-276. For T cells,10⁶ cells were analysed on day 12 after restimulation. Primer sequenceswere: Granzyme A, (forward) 5′ ctctggtccccggggccatc 3′ [SEQ ID. NO: 21]and (reverse) 5′ tatgtagtgagccccaagaa 3′ [SEQ ID. NO: 22]; for GranzymeB, (forward) 5′ ctccacgtgctttcaccaaa 3′ [SEQ ID. NO: 23] and (reverse)5′ ggaaaatagtacagagaggca 3′ [SEQ ID. NO: 24]; for β-actin, (forward) 5′cattgtgatggactccggagacgg 3′ [SEQ ID. NO: 25] and (reverse) 5′catctcctgctcgaagtctagagc 3′ [SEQ ID. NO: 26]. The annealing temperaturewas 55° C. for 27 cycles.

Detection of transduced B cells in vivo. Mice that received pMND-p21gp75transduced B cells followed by T cell clone transfer were sacrificed andsplenic B cells were purified with CD19 microbeads (Miltenyi Biotec).Five μg total RNA was reverse transcribed for the preparation of cDNA.PCR was carried out on cDNA using retroviral vector-specific primers,(forward) 5′ ccctttatccagccctcactc 3′ [SEQ ID. NO: 27] and (reverse) 5′cctggggactttccacaccc 3′ [SEQ ID. NO: 28]. Annealing temperature was 56°C. for 28 cycles.

Statistical analysis. Non-parametric assays were used for evaluatingdifferences between means (Mann-Whitney U test). For assessing airwayhyperreactivity, the area under the curve was calculated and differencesevaluated by the Mann-Whitney U test.

Example 11 In Vivo-Induced Cytolytic CD4+CD25+ Regulatory T CellsPrevent and Suppress Experimental Asthma.Elicitation of Antigen-SpecificRegulatory T Cells In Vivo

A first cytolytic CD4+CD25+ T cell clone (G121) was obtained from miceimmunised with the synthetic peptide p21-35, encompassing a major T cellepitope of the allergen Der p 2. The frequency of such cytolytic T cellswas extremely low as compared to that of CD4+ effector cells of the samespecificity. Attempts to obtain T cells with the same properties usingalternative adjuvants such as alum were unsuccessful.

This could be due to inefficient peptide presentation due for example torapid degradation and/or inefficient late endosome uptake. The in vitrocapacity of various forms of peptide was tested to induce apoptosis of aB cell line (Wehi cells) used as antigen-presenting cell (APC) by theG121 regulatory T cell clone. Methylation of the two cysteines mayenhance the stability of the peptide (p21-35met). Alternatively,coupling p21-35 to a peptide carrying a known subdominant T cell epitopecould increase late endosome uptake. In the BALB/c mouse, the sequenceencompassing amino-acid residues 830 to 844 of tetanus toxoid representssuch a minor epitope (QYIKANSKFIGITEL, [SEQ ID. NO: 18]). A syntheticpeptide covering amino acids 830-844 of tetanus toxoid, linked by twoglycines to p21-35 was therefore produced(QYIKANSKFIGITELGGCHGSEPCNIHRGKPF, [SEQ ID. NO: 20], and for stabilityreasons, the two cysteines were also methylated (modified peptide, mp21-35). mp 21-35 was 100-fold more efficient in inducing Wehi cellapoptosis in the presence of the G121 Treg, as compared to p21-35 (FIG.11 a). Methylation of the 2 cysteine residues of p21-35 (p21-35met) alsoincreased MHC class II presentation, and this stabilised peptide was asefficient as mp 21-35. A control peptide made by reversing the order ofthe two components of mp 21-35 was inefficient at inducing WEHI cellapoptosis.

The methylated form of p21-35 (p21-35met) or the coupled variant (mp21-35) were tested for efficient in vivo elicitation of cytolytic Tcells. BALB/c mice were immunised with Der p 2 in alum. in vitrorestimulation of spleen CD4+ T cells resulted in robust proliferationwhen Der p 2 was used for stimulation but little response to p21-35presentation (FIG. 11 b: saline control group). Subsequently, thecapacity of mp 21-35 to prevent CD4+ T cell proliferation bypre-immunisation of BALB/c mice was tested with either mp 21-35 or amixture of p830-844 and p21-35met, namely the two peptide components ofmp 21-35. This was followed by IP administration of Der p 2 in alum, asabove. A significant reduction of Der p 2-induced proliferation wasobserved only when mp 21-35 was used for pre-immunisation (FIG. 11 b).The production of cytokines made by CD4+ spleen cells stimulated withDer p 2 was identical in the control group and in the grouppre-immunised with the two separate peptide components (FIG. 11 c),showing a Th2-like response. In the group pre-treated with mp 21-35,cytokine production was reduced to undetectable concentrations.

Noteworthy, in vitro stimulation of CD4+ splenocytes obtained from micepre-treated with either mp 21-35 or the mixture of peptides elicited noproliferation to p830-844 (FIG. 11 b), or cytokine production,indicating that the tetanus toxoid-derived peptide did not interferewith the generation of Tregs.

mp 21-35 had to be processed for efficient presentation, as shown byassessing activation of a p21-35 effector T cell (G221N) in the presenceof various inhibitors (FIG. 11 d). This shows that p21-35met, mp 21-35and Der p 2 required internalisation into antigen-presenting cells(inhibited by NaN3/deoxyglucose), fusion with and acidification of thelate endosomes (inhibited by colchicine and chloroquine, respectively).Peptide processing was not required for p21-35met, as shown by absenceof inhibition by addition of leupeptin, a serine/cysteine proteaseinhibitor, reflecting the capacity of MHC class II molecules toaccommodate sequences of up to 15 amino acids.

Taken together, these data indicate that mp 21-35 was efficientlyprocessed in vivo, by contrast to a mixture of its two components, andthat pre-immunisation with mp 21-35 prevented allergen-specific T cellactivation. Based on findings showing that regulation can be morereadily achieved with analogues of T cell epitopes, the capacity ofvarious single amino acid p21-35 mutants to elicit CD4+ T cell after IPimmunisation in alum was determined. In particular, a mutant peptidewith an Ile28Asn mutation, a position known to correspond to the P4 MHCclass II anchoring residue, showed only slightly reduced capacity toinduce T cell proliferation. The mutated form of mp 21-35 (mp 21-35Asn)was therefore use in the remainder of the experiments in this Example.

Derivatisation and Phenotypic Characterisation of TREG Clones

T cell clones were derived for phenotypic analysis from the spleen ofmice injected with either mp 21-35Asn in alum, mp 21-35Asn in CFA/IFA(complete or incomplete Freund adjuvant), Der p 2 or saline as acontrol. A total of 17 clones were obtained, which expansion was fullydependent of addition of IL-2. These clones were maintained at rest for10 days before assessing expression of CD25. Positive clones were thenscreened for their capacity to induce apoptosis of p21-35-loaded Wehicells using annexin V binding as readout. All CD4+CD25+ T cell clones(8/8) derived from mice immunised with mp 21-35Asn in alum were shown tobe cytolytic, while no such clones were obtained with mp 21-35Asn inCFA/IFA (0/5) or from mice immunised with Der p 2 (0/4). Interestingly,a small number of CD4+ effector T cells were obtained from non-immunisedmice (see below) but none were cytolytic.

The phenotype of cytolytic Tregs was characterised. Similar results wereobtained for a total of 15 clones obtained from separate immunisations.Representative Facs results for surface markers are shown in FIG. 12 a(one representative clone) and in Table 5 (4 clones), indicatinghomogenous expression levels.

TABLE 5 Presence of surface markers on 4 cytolytic clones obtained withpeptide mp21-35Asn (expressed as MFI) CD28 CD26L CD103 CD45RB ICOS GITRCTLA-4 T1 5 36 21 6 251 127 96 T2 12 27 25 10 245 150 110 T3 4 39 27 6263 110 92 T4 4 36 14 7 253 129 87

All clones expressed high levels or CTLA-4, CITR and ICOS, withsignificant, though low expression of CD62L and CD103, and hardlydetectable expression of the chemokine receptor CCR7. T-bet wasuniformly expressed but not Foxp3, whilst CD127 was only faint (FIG. 12b). In addition, Treg clones express high levels of Granzyme B andperforin. RT-PCR experiments show detectable levels of Granzyme A mRNA,but at a much lower level than Granzyme B (FIG. 12 c). The cytokinesecretion pattern showed almost exclusively IFN-gamma, but no or verylittle IL-10 and no TGF-β (FIG. 12 d). Surface-bound TGF-β was notdetected.

All cytolytic Tregs were anergic as they did not respond to antigenactivation in the absence of added IL-2. The latter reversed anergywithout restoring IL-2 transcription and with no loss of regulatoryproperties, as shown after restimulation cycles. This suggest anepigenetic alteration, possibly related to hyper-expression of T-bet. Inaddition, these clones expressed high levels of egr-2, known to activatethe transcription of cell cycle negative regulators.

These cells expressed high levels of CD44, but showed low expression ofCD45RB (and CD62L as mentioned above), identifying them as memory cells.Absence of production of suppressive cytokines suggests that theirmechanism of action requires cell-cell contact. Expression of granzymesand perforin ranked such clones among T cells with a cytolyticpotential. Lastly, the Vβ usage of cytolytic Tregs was determined, whichindicates a predominance of Vβ8.1, with some clones belonging to the Vβ7family. The beta chain of a number of clones was sequenced, showingsignificant differences in CDR3, which indicated that the responsetowards the p21-35 peptide was oligoclonal. Treg clones were thereforeevaluated functionally to determine both the mechanism of target celllysis and their capacity to elicit bystander T cell suppression.

Induction of Apoptosis in Antigen Presenting Cells

Lysis of antigen-presenting cells was obtained with both B cells anddendritic cells. Splenic B cells from naïve BALB/c mice, preloaded withp21-35 were induced to caspase 3 activation only when incubated with theR3TB7 cytolytic T cell clone (i.e. another T cell clone with cytolyticactivity, obtained in a similar way) (FIG. 13 a: left panel), but notwhen a control CD4+ effector p21-35-specific T cell was added (FIG. 13 aright panel). The same experiments were repeated using Annexin V as anapoptosis marker, providing the same results. Experiments were carriedout with p21-35-loaded CD11c+ dendritic (FIG. 13 b: left panel) or WEHIcells (FIG. 13 b: right panel). Upon incubation with R3TB7 virtually alldendritic cells (DC) or Wehi cells were induced into apoptosis asmeasured by caspase 3 activation. The same experiments were carried outwith Annexin V and showed identical results.

Apoptosis can be induced by either the Fas-FasL pathway or by secretionof cytotoxic granules containing granzymes. Attempts were made toinhibit each of these pathways. Addition of increasing concentrations ofan antibody towards FasL, or of granzyme B inhibitors, to a cell culturecontaining p21-35 loaded WEHI cells and a cytolytic T cell clone,increased the number of surviving WEHI cells in a dose-dependent manner(FIG. 13 c). In a number of experiments it was shown that the anti-FasLantibody restored up to 80% of WEHI cell survival. Only partialrestoration of survival was obtained with granzyme B inhibitors and onlywhen high doses, close to cell cytotoxicity, were used, indicating thatgranzyme B did not account for much of the cell cytolysis. In additionalexperiments, EGTA was used as an inhibitor of granule exocytosis, whichalso showed only partial restoration of Wehi cell survival.

It was further evaluated whether target cell lysis required contactbetween cells. To this end, CD11+ DC loaded with p21-35 were used (or mp21-35Asn in parallel experiments). When loaded DC cells were incubatedwith the G121 cytolytic T cell clone, lysis was observed in 89% of thecells as measured by Annexin V expression (FIG. 13 d, left panel). Whenthe same experiments were carried out in a transwell system, DC lysiswas limited to 15%, indicating that lysis required direct contactbetween cells (FIG. 13 d, right panel).

To further ascertain that APC lysis required direct contact withcytolytic T cells through MHC class II presentation of p21-35, anexperiment was carried out in which two identical populations of Wehicells were loaded with either p21-35 or with an irrelevant peptide(p71-85). These two populations could be distinguished by differentialCFSE labelling. It can be seen from FIG. 13 e that, while WEHI cellspresenting p21-35 were fully lysed, only 40% of p71-85-loaded cells wereaffected.

Altogether, it emerges that cytolytic T cell clones induced apoptosis ofDC and B cells by a mechanism requiring the formation of an immunesynapse by MHC class II-dependent peptide presentation. Significantparticipation of Fas-FasL is demonstrated, but only limited involvementof granzyme B.

Bystander T Cell Suppression

The mechanism of bystander T cell suppression was examined withpolyclonal CD4+CD25(−) T cells and with various CD4+ effector T cellclones.

First, the capacity of Tregs was assayed to suppress the proliferationof CD4+CD25(−) T cells after anti-CD3 activation in the presence ofantigen-presenting cells. The results obtained for two cytolytic T cellclones (G121 and R3TB7) is shown in FIG. 14 a. The number of detectableCD4+CD25(−) T cells, as well as the number of observed divisionsdramatically dropped within 48 h incubation when either one of the twocytolytic clones were added (upper left and middle panels). This effectwas even more pronounced at 72 h for the second T cell clone (lowermiddle panel). Interestingly, only activated CD4+CD25(−) T cells werelysed, as can be seen from the vertical axis representing blastformation. The control experiment in which Treg was replaced by anidentical number of unlabeled CD4+CD25(−) T cells (right panels)eliminated a possible artefact related to variable numbers of totalcells in the culture medium.

Next, the kinetics of suppression were analysed. In the experimentsshown in FIG. 14 b, it can be seen that 48 and 70% of CFSE-labelledCD4+CD25(−) T cells express Annexin V after 18 and 24 h co-incubationwith a cytolytic Treg (R3TB7), respectively, using the same assay systemas in FIG. 14 a.

Such a rapid effect suggests the involvement of secretory pathwayssimilar to those used by CD8+ cytotoxic T cells. The experiments weretherefore repeated to verify whether inhibition of granules exocytosisby addition of EGTA to the culture would inhibit suppression. Basicallyno inhibition of the suppression of bystander T cells examined at 72 hwith 2 concentrations of EGTA (FIG. 14 c).

Next, it was evaluated whether T cell clones of defined specificity anddifferent phenotypes could be suppressed by cytolytic T cells whenactivated by cognate recognition of corresponding antigens, instead ofnon-specific anti-CD3 activation. To this end, three clones werederivatisised from BALB/c mice, namely a Th2 cell specific for a secondunrelated allergen from the same source (Der p 1), a Th1 cell specificfor a second major T cell epitope of Der p 2 (amino-acids 71-85) and aTh0 clone specific for the 830-844 subdominant T cell epitope of tetanustoxoid. Each of these 3 clones was used in all the assay systemsreported here. Results are shown for one single clone in each assay, butresults were confirmed in all possible combinations of assays andclones.

In a first set of assays, the proliferation of a T cell clone wasmeasured using CFSE-labelled cells after presentation of thecorresponding antigen by T cell depleted splenocytes. To eliminate anartefact due to competition for nutrient in the culture medium, an equalquantity of the unlabeled Th2 clone was added to the CFSE-labelled Th2clone. A Th2 clone specific for Der p 1 readily proliferated as measuredover a time period of 72 h (FIG. 14 d). In parallel assays, APC werealso loaded with p21-35 and a cytolytic T cell clone added at a 1/1ratio to the Der p 1 specific Th2 clone. The Figure shows that only 5%of the Th2 clone survived after 72 hours incubation. The Figure alsoshows that addition of specific antibodies to FasL over the entireincubation period resulted in a partial inhibition of suppression (36%residual Th2 clone), whilst an anti-GITR or anti-Lag3 antibody had noeffect. Identical results were obtained with a Th1 clone to Der p 2 andwith a Th0 clone to tetanus toxoid. The clones were therefore amenableto suppression by cytolytic T cells independently of their maturationstate, provided activation occurred through MHC class II cognateinteraction.

The question as to whether contact interaction was required between thecytolytic T cells and bystander T cells was examined in a transwellculture system. Results are shown in FIG. 14 e. T cell depletedsplenocytes loaded with both p21-35 and p71-85 sustained theproliferation of a p71-85 specific Th1 clone. Addition of a p21-35cytolytic Treg suppressed proliferation (middle panel). Whenpeptide-loaded APC were separated in a transwell culture system and thecytolytic cell line added to one compartment, with the 71-85 specificTh1 clone in the second compartment, no suppression was observed (rightpanel). It could therefore be concluded that suppression was notmediated by soluble factors. These experiments were confirmed using theTh2 cell clone to Der p 1.

Cytolytic Tregs efficiently prevented bystander T cells blast formation.The average cell size of bystander T cells is only marginally increasedwhen co-cultured in the presence of activated cytolytic Treg (R3TB7) asshown in FIG. 14 f. This was accompanied with an increased proportion ofcell death as measured with the CFSE-labelled bystander cells. Thepercentage of cell death was 26% for bystander cells alone, 36% with thecontrol p830-844 clone used in place of the cytolytic clone, 34% withthe cytolytic clone without activation, and 51% with the cytolytic clonein the presence of the activating peptide.

In order to exclude that bystander suppression could be partly due toartefacts, due to the lysis of APC, additional assays were carried outin which two distinct populations of APC (Wehi-231 B cells), eachincubated with either p21-35 or with a readout peptide (the Der p 1derived T cell epitope or p830-844 of tetanus toxoid). The two APC werecultured in the same well and both a cytolytic T cell clone and aCFSE-labelled effector cell. It was observed that the labelled T cellclone was suppressed. APC presentation of p21-35, required to activatecytolytic Tregs, was replaced by a combination of anti-CD3 and anti-CD28antibodies. Under such circumstances the proliferation of effector cellsactivated by cognate recognition was also suppressed. Taken togetherwith experiments reported in FIG. 14 e, this shows that cytolytic T cellclones suppressed bystander T cells in the absence of APC apoptosisinduction.

Cytolytic TREGS Lyse P21-35-Loaded B Cells In Vivo

Cytolytic Tregs induce apoptosis of APC in vitro. To determine whetherthis activity was relevant in an in vivo setting, transgenic B cellsexpressing p21-35 in the context of MHC class II determinants were used.Such transgenic B cells persist for at least 3 months in the spleenafter adoptive transfer. First, it was verified whether cytolytic Tregscould induce apoptosis of transgenic B cells in vitro. 52% of B cellsare induced in apoptosis after 18 h incubation with a cytolytic T cellclone (FIG. 15 a). Expression of the construct in spleens of controlmice (lanes B), but not in spleens of mice transferred with bothtransgenic B cells and Tregs (lanes A) is shown in FIG. 15 b. No viralconstruct was detected in lung cells, in the absence of allergenexposure. These data indicated that cytolytic T cells maintained theircapacity to lyse B cells presenting the cognate antigen after in vivotransfer.

Cytolytic Tregs Accumulate into the Lungs Upon Allergen Exposure

The memory phenotype of the Tregs, combined to the absence of CCR7 andlow levels of CD103, suggest that they can migrate to the lungs, wherethey can exert their suppressive activity.

Two different cytolytic T cell lines were labelled with either CFSE orSNARF (both markers for cytoplasmic proteins, emitting at differentwavelengths) and used in separate experiments. Accumulation ofCFSE-labelled cells was found in perivascular and peribronchial lungareas (data not shown). Control mice receiving labelled Tregs but noallergen instillations showed virtually no lung fluorescence.

To establish whether Tregs represent a significant proportion oflymphocytes accumulating in lungs, and to discard a possible artefactrelated to the inherent toxicity of fluorescence labelling, unlabeledTregs were adoptively transferred to BALB/c mice. In these experiments,use was made of the fact that cytolytic cells expressed V138.1 andcounted the proportion of positive cells over the entire CD4+ populationaccumulating in lungs after nasal instillation with allergen. Under suchconditions, it is known that very little lymphocytes are attracted tolungs in the Der p 2 model of asthma. In the group of mice transferredwith the cytolytic T cell clone more than 90% of CD4+ cells expressedV138.1, whilst only 25 and 20% of such cells were detected in the grouptreated with the control vβ8.1.+ cell line, and in a control group ofmice that received Der p 2 by inhalation but no T cells, respectively(FIG. 15 c).

It is therefore concluded that cytolytic Tregs accumulated into thelungs upon allergen challenge by nasal instillation.

Cytolytic Treg Clones Prevent and Suppress Experimental Asthma

The above-described experiments suggested that cytolytic T cell clonescould be of value for the control of specific immune responses in vivo.The requirement for MHC class II cognate interaction and induction ofapoptosis of APC could provide an opportunity to suppress the entireresponse towards single antigens. In addition, their accumulation inlung tissue could favour their regulatory activity on some of thefeatures associated with asthma.

This was tested by adoptively transferring cytolytic T cells specificfor p21-35 in both a preventive and suppressive settings. Theexperimental asthma model to Der p 2 was used as described above. Twocytolytic T cell lines were used, as obtained from BALB/c mice immunisedwith mp 21-35Asn.

The results of the prevention experiments are shown in FIG. 16( a-f). Itcan be seen that the transfer of cytolytic T cells prior to induction ofDer p 2 sensitisation reduced the number of cells recovered from BALF tovalues observed in naïve animals (not shown in FIG. 16 a), withvirtually no cells except macrophages. The control cell line had noeffect on BALF cell recovery as compared to the positive control group.The two cytolytic cell clones produced essentially the same effect. Theproduction of TGF-β in BALF was undetectable with the two Tregs, whileIL-10 production was suppressed with the second clone. Significantreduction of lung eosinophil infiltration and goblet cell hyperplasiawas observed. In addition, airway hyper-reactivity measured byinhalation of increasing doses of methacholine was practically reducedto levels observed in naïve mice (naive).

When the same two cytolytic cell lines were used after Der p 2sensitisation, similar improvement of biologic and functional parameterswere observed (FIG. 16( g-l)). One notable exception, however, was thepresence of goblet cell hyperplasia. Interestingly, airway reactivity tomethacholine was comparable to that of naïve animals. In additionaltesting these last results were verified by dynamic compliancemeasurements, which provided essentially the same results.

Example 12 Immunisation of BALB/c Mice with Der p 1 Derived Peptide

Two groups of BALB/c mice were immunised by the subcutaneous route witheither 20 μg of peptide with a T cell epitope sequence of the allergenDer p1 (SNYCQIYPPNANKIR [SEQ ID. NO: 5]) or a modified version thereofCGFS SNYCQIYPPNANKIR [SEQ ID. NO: 7] having at the N-terminus thesequence CGFS [SEQ ID. NO: 6].

The amino acids of Der p 1 which reside in the cleft are S114 (or N115)to P122 (or P123) (where MHC class II haplotypes binding 9 amino acidsin the cleft are concerned). The SNYC sequence which is present in theDer p 1 is thus not accessible and can not interact with other proteinsand perform its reducing activity.

After 3 injections, mice were sacrificed and CD4+ T cells were purifiedfrom the spleen and cloned by limiting dilution. T cell clones obtainedfrom mice immunised with the SNYCQIYPPNANKIR [SEQ ID. NO: 5] peptideproduced effector CD4+ T cells characterised by proliferation andcytokine secretion following cognate interaction with MHC class IIpresentation of peptide. Mice immunised with the peptide CGFSSNYCQIYPPNANKIR [SEQ ID. NO: 7] produced T cell clones with cytolyticproperties, as determined in an assay similar to that described inExample 10.

To determine whether T cells with cytolytic properties (as shown inExample 10) had the capacity to induce apoptosis of effector T cells,antigen-presenting cells (APC) were prepared from adherent spleniccells. APCs were incubated with the Der p 1 allergen for presentationinto MHC class II determinants.

As shown in FIG. 17, CD4 effector cells readily proliferated when addedto antigen-loaded APCs. Addition of cytolytic T cells induced death ofCD4 effector cells (7-AAD positive staining (FL3-H)). FIG. 17 showsapoptosis in 73% of the effector cells and the strong abrogation of theproliferation of CD4 effector cells.

Example 13 Adoptive Transfer of T Cell Clones with Cytolytic ActivityFully Prevents and Suppresses Asthma Elicited by Nasal Instillation ofthe Der p 1 Allergen

BALB/c mice were submitted to 2 series of 3 daily nasal instillationsseparated by 1 week, using 100 μg of the Der p 1 allergen. The day afterthe last nasal instillation, mice are sacrificed and checked for thepresence of alterations characteristic of allergic asthma in thebronchoalveolar space and lung.

Two additional groups of mice are adoptively transferred with cytolyticT cell clones obtained as described in Example 11 using the Der p Ipeptide with the motif, either prior to or after the first series ofnasal instillations.

BALF differential cell counts were carried out 4 days after the lastseries of nasal instillations (6 BALB/c per group). Cells were obtainedby bronchoalveolar lavage of lungs and identified on cytospins asmacrophages, neutrophils, eosinophils or lymphocytes. Mice received 2series of 3 nasal instillations with either 100 μg Der p 1 (model) orNaCl (negative). FIGS. 18A and B show that when cytolytic T cells areadministered before or after the first nasal instillation series, thisresults in complete abolition of eosinophil infiltration into thebronchi. As can be seen, the total number of cells recovered in thelavage fluid is within the range of what is obtained from naïve animals.

Broncho-alveolar lavage fluids were tested for the presence of cytokines(Table 6). Mice receiving a cytotoxic clone showed very low cytokinerecovery, including the concentration of IL-10 which is significantlyincreased in the model.

TABLE 6 BAL cytokines determination of mice treated with Der p 1modified peptide [SEQ ID. NO: 7] (three days after the last nasalinstillation). TNF- IFN- TGF- alpha gamma IL-5 IL-4 IL-2 IL-10 IL-13beta Prevention 0.7 0 0.3 0 0.7 10 0.2 5.8 Suppression 0 0 0.5 0.7 0.8 20.3 0 Model 0 0 0 0 0 44 4 1 Neg. 6 1 0.5 2 0 0 0 0 Results representaverage concentrations obtained from 6 BALB/c (pg/ml).

Example 14 Effect of Increasing the Redox Potency of Modified Peptideson the Capacity to Activate Cytolytic Regulatory T Cells

Antigen-presenting cells were loaded with p21-35 in its nativeconfiguration [SEQ ID. NO: 2] or p21-35 in which serine in position 24is substituted by cysteine CHGCEPCIIHRGKPF [SEQ ID. NO: 29]. Thissubstitution creates a redox moiety of the type C-x-x-C.

Several regulatory T cell clones with cytolytic activity were incubatedwith APC. FIG. 19 shows that the peptide carrying the C-x-x-C redoxsequence induces a higher degree of T cell activation, assessed bycytokine production, than its C-x-x-S counterpart. It is also shown thatthe C-x-x-C sequence induces the transcription of mRNA for both FasL andgranzymes, more than its C-x-x-S counterparts. The granzymes are two ofthe key players in induction of apoptosis of target cells.

Example 15 Prevention and Suppression of Beta-Lactoglobulin Allergy

Bovine beta-lactoglobulin (BLG) is a major allergen in human milkallergy. A mouse model is used to determine whether modified peptidescould alter the specific response towards BLG. The peptide CHGCAQKKIIAEK [SEQ ID. NO: 30] encompassing the thioredox motif sequenceC-H-G-C [SEQ ID. NO: 31] linked to a T cell epitope of BLG issynthesised.

BALB/c mice are immunised by 2 SC injections with 20 μg of the peptideof sequence CHGC AQKKIIAEK [SEQ ID. NO: 30] in alum. This is followed 15days later by intraperitoneal sensitisation to BLG (5 μg in alum), on 2occasions at a fortnight interval. Alternatively, peptide administrationis given 15 days after IP sensitisation to BLG.

Hypersensitivity to BLG is verified in the mouse by assessing bronchialhyperreactivity after intranasal administration of BLG. All mice aresubmitted to intranasal administration of 10 μg of BLG in saline 10 daysafter the last injections. A control group is included in whichintraperitoneal sensitisation is carried out without peptideimmunisation.

Mice injected with the modified peptide epitope prior to or aftersensitisation have completely lost the capacity to react to BLG nasalinstillation. This is shown by the lack of eosinophils inbronchoalveolar lavage fluid and the absence of hyperreactivity uponchallenge with increasing doses of methacholine, as compared to thecontrol group, in which both eosinophils and hyperreactivity areobserved.

Example 16 Prevention and Suppression of Multiple Sclerosis

Groups of C57BL/6 mice are immunised subcutaneously (20 μg) with peptide(CHGS YRSPFSRVVHLYR [SEQ ID. NO: 32], which contains the sequence motifC—X(2)-S) or control peptide (YRSPFSRVVHLYR [SEQ ID. NO: 33]) adsorbedonto aluminium hydroxide. Three injections are performed at 2-weekintervals. Ten days after the last immunisation, mice are sacrificed andCD4+ T cells (2×10⁶ cells) are prepared from the spleen using magneticbeads. CD4+ T cells are then stimulated in vitro by the MOG T cellepitope (20 μg/ml) presented by adherent spleen cells (2×10⁶ cells).

After four re-stimulations, a T cell line is tested in a bystandersuppression assay with, as target cells, polyclonal CD4+CD25− cellsobtained from animals in which EAE (Experimental autoimmuneencephalomyelitis) was effective. Only the cells obtained from animalsimmunised with the peptide containing the C—X(2)-S sequence motif havethe capacity to induce death in target cells, as compared to the controlconsisting in effector CD4+CD25− from EAE animals, as shown in FIG. 20.

A group of C57BL/6 mice is adoptively transferred with a CD4+MOG-specific regulatory T cell clone followed after 1 day by a protocolmeant to induce a multiple sclerosis-like syndrome. This involvesadministration of the MOG peptide in complete Freund's adjuvant and 2injections of Pertussis toxin. This protocol elicits an expansion of theeffector T cell clone, which results in the development of signscompatible with multiple sclerosis within 12 days after the MOG peptideadministration. It is observed that the clinical score developed by micepre-treated with the cytolytic T cell clone is significantly reduced ascompared to mice receiving only the full protocol of disease induction(FIG. 21).

It is observed that the clinical score developed by mice pre-treatedwith the cytolytic T cell clone is significantly reduced as compared tomice receiving only the effector T cell clone, as shown in FIG. 21.

Example 17 Prevention of Multiple Sclerosis by Peptide Immunisation

In the model group, 3 C57BU6 mice received, at day 0, SC injection of100 μg MOG peptide/400 μg Mycobacterium butyricum in CFA and ipinjection of 300 ng Bortetella pertussis in NaCl. At day +2, a secondinjection of B. pertussis was given.

In the prevention group, 5 C57BL/6 mice are immunised by 5 injectionswith 20 μg CSMOG peptide (CHGS YRSPFSRVVHLYR [SEQ ID. NO: 32]), whichcontains the sequence motif C—X(2)-S, in IFA at 14 days interval beforedisease induction as in the model group. Control experiments areperformed with the peptide [SEQ ID. NO: 33] lacking the motif at itsN-terminus.

Scores were established as 0: no disease, 1: limp tail, 2: limp tail andloss of weight higher than 10%, 3: partial paralysis of hind limbs.

FIG. 22 shows that pre-treatment with modified peptide [SEQ ID. NO: 32]completely abolishes the development of the syndrome.

Example 18 Prevention and Suppression of Spontaneous Insulin-DependentDiabetes with GAD65 Derived Peptides

Non-obese diabetes (NOD) mice constitutes a suitable animal model forspontaneous insulin-dependent diabetes.

In such animals, as in human beings, an early immune response to theautoantigen glutamic acid decarboxylase (GAD65) is observed at a time atwhich insulitis can be seen, from which the response extends byintramolecular and intermolecular spreading. Inducing tolerance to GADby administration of the protein to neonates prevents the onset ofdiabetes.

The carboxy-terminal region of GAD65, and in particular the fragment524-543 SRLSK₅₂₈VAPVIKARMMEYGTT [SEQ ID. NO: 34], is recognised byspecific T cells. Some of such T cells are pathogenic, such as thoserecognising fragment 530-543, whilst others do not elicit the disease,such as those recognising the fragment 524-538.

Lys528 constitutes a P1 anchoring residue. For the generation of apeptide in accordance with the invention, Serine residues P−4 and P−1are replaced by a cysteine, which results in CRLC KVAPVIKARMM [SEQ ID.NO: 35].

Treg cells with cytotoxic properties are elicited by immunisation withthe above modified peptide comprising the T cell epitope of the GAD65protein. T cells obtained from the spleen of NOD mice aged 20 weeks, ata time at which insulitis is present as well as overt diabetes areexpanded in vitro with peptide 524-543 in order to generate pathogenic Tcell clones.

NOD mice are immunised with peptide containing the thioredoxin consensussequence in IFA (incomplete Freund adjuvans) from the age of 2 weeks. Tcells are expanded in vitro to generate clones with regulatoryproperties.

Polyclonal cells purified from mice immunised with the thioredoxinconsensus sequence induce apoptosis in polyclonal CD4 cells obtainedfrom peptide 524-543 immunised NOD mice when stimulated byantigen-presenting cells loaded with peptide 524-543 (see FIG. 23). Thetable in FIG. 23 represents the percentage of double positive cells(dead cells) after substraction of background values obtained withoutthe regulatory population.

Adoptive transfer of Treg at the age of 2 weeks in NOD mice, namelybefore the onset of insulitis (3 weeks of age) is shown to fully preventthe onset of diabetes.

Direct immunisation of NOD mice with the peptide CRLC KVAPVIKARMM [SEQID. NO: 35]. from the age of 2 weeks is shown to fully prevent diabetesand lesions of insulitis.

In a suppressive setting, adoptive transfer of Treg at different agesbetween 6 and 20 weeks is shown to suppress diabetes and insulitis inNOD mice. Immunisation with peptide CRLC KVAPVIKARMM [SEQ ID. NO: 35]after the age at which insulitis is already prominent (15 weeks of age)is shown to suppress diabetes and insulitis.

Example 19 Prevention and Suppression of Spontaneous Insulin-DependentDiabetes with Insulin Derived Peptides

Tregs with cytolytic properties are elicited by immunising NOD mice withthe T cell epitope of insulin linked to the motif C—X(2)-C with andwithout a glycine linker.

The peptides which are synthesised are:

EALYVCGERG CGPC [SEQ ID. NO: 36] EALYVCGERG G CGPC [SEQ ID. NO: 37]EALYVCGERG GG CGPC [SEQ ID. NO: 38] EALYVCGERG GGG CGPC [SEQ ID. NO: 39]EALYVCGERG GGGG CGPC [SEQ ID. NO: 40]

The Treg cells obtained with these peptides induce apoptosis ofinsulin-specific pathogenic T cells in an in vitro system in which bothpathogenic and regulatory cells are activated by presentation ofinsulin. They further prevent or suppress the onset of diabetes andinsulitis after adoptive transfer of Tregs prior to (2 weeks of age) orafter (6 weeks of age) the spontaneous onset of insulitis, respectively.

The Treg cells obtained with these peptides also prevent or suppress theonset of diabetes and insulitis when elicited in vivo upon immunisationwith peptide of sequence containing the thioredoxin consensus sequence,starting at the age of 2 weeks for prevention or 15 weeks forsuppression, respectively.

Example 20 Prevention and Suppression of Autoimmune Thyroiditis withThyroperoxidase Derived Peptides

Autoimmune thyroiditis in man is associated with the production ofantibodies to thyroid peroxidase. Antibodies and specific T cells to TPOresult in thyroid cell destruction by a combination of cytotoxic andcytolytic mechanisms, leading to hypothyroidism. On purpose immunisationof C57BI/6 mice with TPO elicits a disease state identical to humanpathology and is therefore considered as a suitable model for autoimmunethyroiditis.

The fragment 540-559 (QGQLM₅₄₄NEELTERLFVLSNV [SEQ ID. NO: 41] of TPOencompasses a dominant T cell epitope of TPO recognised by C57BI/6 mice.

The use of different algorithms identified residue Met544 as the firstanchoring residue into MHC-class II determinants. Amino acids P−4 to P−1are replaced by the sequence of D-isomer CGPC [SEQ ID. NO: 42], whichgenerates the sequence (CGPC)_(D-isomer) MNEELTERL [SEQ ID. NO: 43].

C57BI/6 mice are immunised twice with the experimental peptide [SEQ ID.NO: 43] or the control peptide [SEQ ID. NO: 41] or QGQLM₅₄₄NEELTERL [SEQID. NO 48] in CFA/IFA. Ten days after the last immunisation, mice aresacrificed and T cells are prepared from the spleen. CD4+ T cells areexpanded in vitro using antigen-presenting cells loaded with the 540-559sequence. T cell clones are obtained by limiting dilution.

The capacity of T cell clones generated with the experimental peptide(CGPC)_(D-isomer) MNEELTERL [SEQ ID. NO: 43] to suppress activation ofeffector T cells obtained by immunisation with peptide of sequence540-559 is tested in vitro. Herein, antigen-presenting cells are loadedwith peptide 540-559. Addition of the effector T cell clone results inactivation and proliferation of such cells, as measured by thymidineincorporation. When a T cell clone with regulatory activity is added tothe system, together with the effector clone, activation andproliferation of the latter is completely inhibited.

Adoptive transfer of regulatory T cells to C57BI/6 mice prior to orafter immunisation with peptide 540-559 prevented or suppressed,respectively, induction of thyroiditis, as evaluated by assessinglymphocytic infiltration of the thyroid.

Example 21 Prevention and Suppression of Autoimmune Thyroiditis withThyroglobulin Derived Peptides

An immune response towards thyroglobulin is a common feature of humanautoimmune thyroiditis. Induction of such a response in experimentalthyroiditis upon injection of peptides encompassing T cell epitopes hasbeen obtained in genetically predisposed animals such as H2k mice.

The thyroglobulin fragment 2340-2359 (QVA₂₃₄₂ALTWVQTHIRGFGGDPR [SEQ ID.NO: 44]) encompasses a dominant T cell epitope of thyroglobulinrecognised by AKR/J mice. The use of different algorithms identifiesresidue Ala2342 as the first anchoring residue into MHC-class IIdeterminants. Amino acids P−4 to P−1 were replaced by the sequence CGPS[SEQ ID. NO: 13], which generates the sequence CGPS AALTWVQTH [SEQ ID.NO: 45].

AKR/J mice are immunised twice with the experimental peptide [SEQ ID.NO: 45] and the control peptide LDQVAALTWVQTH [SEQ ID. NO: 49] inCFA/IFA. Ten days after the last immunisation, mice are sacrificed and Tcells prepared from the spleen. CD4+ T cells are expanded in vitro usingantigen-presenting cells loaded with the 2340-2359 fragment. T cellclones were obtained by limiting dilution.

The capacity of T cell clones generated with peptide CGPS AALTWVQTH [SEQID. NO: 45] to suppress activation of effector T cells obtained byimmunisation with peptide of sequence 2340-2359 is tested in vitro.Herefore, antigen-presenting cells were loaded with peptide 2340-2359.Addition of the effector T cell clone resulted in activation andproliferation of such cells, as measured by thymidine incorporation.When a T cell clone with regulatory activity is added to the system,together with the effector clone, activation and proliferation of thelatter is completely inhibited.

Adoptive transfer of regulatory T cells to AKR/j mice prior to or afterimmunisation with peptide 2340-2359 prevents or suppresses,respectively, induction of thyroiditis, as evaluated by assessinglymphocytic infiltration of the thyroid.

Example 22 Prevention and Suppression of Pollen Allergy with BirchPollen Allergen Derived Peptides

Sensitivity to birch pollen is a common cause of rhinitis and asthma.However, about 60% of birch pollen-sensitised subjects develop symptomsupon ingestion of fruits of the Rosacea family, such as apple, pears,plums and cherries. Cross-reactivity between Bet v 1 (the main birchpollen allergen) and such food has been demonstrated at both the levelof specific IgE antibodies and T cells. In particular a T cell epitopelocated at the carboxy-terminal end of the Bet v 1 molecule, which isconserved between various isoforms, shown a high degree of homology withthe sequence of an equivalent allergen of apple (Mal d 1). Effector Tcells recognising fragment 142-156 of Bet v 1 are strongly activatedwhen exposed to the Mal d 1 corresponding epitope.

The peptide LRAVESYLLAH [SEQ ID. NO: 46] corresponding to residues144-154 of Bet v 1, and containing a T cell epitope, is modified byaddition of the sequence (CGPC)_(D-isomer) [SEQ ID. NO: 42], at itsaminoterminal end resulting in (CGPC)_(D-isomer) LRAVESYLLAH [SEQ ID.NO: 47].

This peptide is adsorbed on aluminium hydroxide using 50 μg of peptidefor 1 mg of alum. Three SC injections of 50 μg of peptide were carriedout at 2-week intervals. Two weeks after the last injection, blood isdrawn from a peripheral vein and CD4+ T cells purified by cell sortingon magnetic beads. CD4+ T cells were added to culture medium in whichHistocompatible dendritic cells, used as antigen-presenting cells, wereincubated with either the Bet v 1 antigen, or with tetanus toxoid as acontrol, for 2 h at room temperature. The cells were then washed andpurified CD4+ T cells were added to the culture medium. Such CD4+ Tcells induced apoptosis of dendritic cells presenting the Bet v 1antigen, but not of dendritic cells presenting the tetanus toxoidprotein. Incubation of CD4+ T cells with dendritic cells loaded with theMal d 1 antigen resulted in dendritic cell apoptosis.

Patients presenting with allergic symptoms upon exposure to birch pollentogether with an oro-pharyngeal allergy to ingestion of apple andtreated by 3 SC injections of peptide CGPC LRAVESYLLAH [SEQ ID. NO: 47]failed to react to either exposure to pollen or contact with apple ontothe oro-pharyngeal mucosa.

Vaccination with a peptide containing a T cell epitope from the allergenBet v 1 from birch pollen modified as to contain a redox moiety elicitscytolytic regulatory T cells that eliminate symptoms related to birchpollen exposure and those resulting from ingestion of fruits carrying ahomologous T cell epitope.

1-36. (canceled)
 37. An isolated immunogenic peptide derived from anantigenic protein comprising an artificial sequence comprising: a T cellepitope of said antigenic protein; and motif C—X(2)-[CST] or[CST]-X(2)-C, said motif being either adjacent to said epitope, orseparated from said epitope by a linker of at most 7 amino acids, 38.The isolated immunogenic peptide according to claim 37 wherein saidmotif does not naturally occur within a region of 11 amino acidsN-terminally or C-terminally of the T-cell epitope in said protein. 39.The isolated immunogenic peptide according to claim 37 wherein the motifis C—X(2)-C, said motif being either adjacent to said epitope, orseparated from said epitope, by a linker of at most 7 amino acids. 40.The isolated immunogenic peptide according to claim 37 wherein the motifis C—X(2)-[ST] or [ST]-X(2)-C, said motif being either adjacent to saidepitope, or separated from said epitope, by a linker of at most 7 aminoacids, wherein, if said motif is C—X(2)-S or S—X(2)-C, said T cellepitope does not comprise the sequence EPCIIHRGKP (SEQ ID. NO: 1) of thep21-35 peptide of Der p
 2. 41. The isolated immunogenic peptideaccording to claim 40, wherein, if said motif is C—X(2)-S or S—X(2)-C,said antigenic protein is not Der p
 2. 42. The isolated immunogenicpeptide according to claim 37, wherein said artificial sequence has alength of between 12 and 19 amino acids.
 43. The isolated immunogenicpeptide according to claim 37, wherein the antigenic protein is anauto-antigen or an allergen.
 44. A method of treating or preventing anauto-immune disorder in an individual, comprising the steps ofadministering an isolated immunogenic peptide according to claim 37 tosaid individual, wherein the immunogenic peptide is derived from anauto-antigenic protein.
 45. A method of treating or reducing thesymptoms of an allergic condition in an individual, comprising the stepsof administering an isolated immunogenic peptide according to claim 37to said individual, wherein the immunogenic peptide is derived from anallergen.
 46. A method of treating or preventing an allergic conditionor an auto-immune disorder in an individual, comprising the steps of:providing peripheral blood cells of said individual, contacting saidcells with an antigenic peptide according to claim 37, expanding saidcells, and administering said expanded cells to said individual.
 47. Amethod for preparing a peptide of an antigenic protein capable ofeliciting cytolytic CD4+ T cell activity said method comprising thesteps of: providing a peptide sequence consisting of a T-cell epitope ofsaid antigenic protein, and linking to said peptide sequence a sequencecomprising motif C—X(2)-C, such that said motif and said epitope areeither adjacent to each other or separated by a linker of at most 7amino acids.
 48. A method for preparing a peptide of an antigenicprotein capable of eliciting cytolytic CD4+ T cell activity said methodcomprising the steps of: providing a peptide sequence consisting of aT-cell epitope of said antigenic protein, wherein the motif C—X(2)-[CST]or [CST]-X(2)-C does not naturally occur within a region of 11 aminoacids N-terminally or C-terminally of the T-cell epitope in saidprotein, and linking to said peptide sequence a sequence comprisingmotif C—X(2)-[CST] or [CST]-X(2)-C, such that said motif and saidepitope are either adjacent to each other or separated by a linker of atmost 7 amino acids.
 49. A method for preparing a peptide of an antigenicprotein capable of eliciting cytolytic CD4+ T cell activity said methodcomprising the steps of: providing a peptide sequence consisting of aT-cell epitope of said antigenic protein, and linking to said peptidesequence a sequence comprising motif C—X(2)-[CST] or [CST]-X(2)-C, suchthat said motif and said epitope are either adjacent to each other orseparated by a linker of at most 7 amino acids, wherein, where saidmotif is C—X(2)-S or S—X(2)-C said T-cell epitope does not comprise thesequence EPCIIHRGKP (SEQ ID. NO: 1) of the p21-35 peptide of Der p 2.50. The method of claim 49, wherein said antigenic protein is not Der p2.
 51. The method of claim 49, which further comprises modifying thesequence of said peptide by modifying the amino acids in the epitope,thereby ensuring that in said modified peptide the sequence of theepitope is modified such that ability to fit into the MHCII cleft ismaintained.
 52. A method for preparing an isolated immunogenic peptideof an antigenic protein capable of eliciting cytolytic CD4+ T cellactivity said method comprising the steps of: (a) identifying withinsaid antigenic protein a sequence comprising a T cell epitope flanked,in said antigenic protein by motif C—X(2)-[CST] or [CST]-X(2)-C within aregion of 11 amino acids N-terminally or C-terminally of said T-cellepitope; and (b) generating a peptide comprising said sequence as anisolated peptide of between 12 and 19 amino acids, wherein saidantigenic protein is not Der p
 2. 53. The method of claim 52, whichfurther comprises modifying the sequence of said peptide by modifyingthe amino acids in the motif and/or by modifying the number of aminoacids between the motif and the epitope and/or by modifying the epitopesequence, thereby ensuring that in said modified peptide: the ability ofthe T cell epitope to fit into the MHCII cleft is maintained, the motifis conserved, and said motif and said epitope remain adjacent to eachother or separated by a linker of at most 7 amino acids.
 54. The methodaccording to claim 52, which further comprises the step of attaching alate endosomal targeting sequence to the peptide obtained in step (b).55. A method of identifying a population of cytotoxic Tregs whichcomprises determining that, the cells have the followingcharacteristics: the cells express CD4, the cells do not express IL-10or TGF-beta, and the cells express Krox-20 and produce granzymes and Fasligand.
 56. A method of identifying a population of cytotoxic Tregswhich comprises determining one or more of the followingcharacteristics, when compared to non-cytotoxic Tregs: an increasedexpression of surface markers including CD103, CTLA-4, FasL, and ICOSupon activation, a high expression of CD25, expression of CD4, ICOS,CTLA-4, GITR and low or no expression of CD127 (IL7-R), the expressionof transcription factor T-bet and/or egr-2 (Krox-20) but not of thetranscription repressor Foxp3, a high production of IFN-gamma and no oronly trace amounts of IL-10, IL-4, IL-5, IL-13 or TGF-beta, and anincreased expression of markers including FasL and granzymes B and Cupon activation.
 57. The method according to claim 56, which furthercomprises determining that these cells do not respond to the activationby TCR recognition.
 58. A method for obtaining a population ofantigen-specific regulatory T cells with cytotoxic properties, themethod comprising the steps of: providing peripheral blood cells,contacting said cells with an immunogenic peptide according to claim 37,and expanding said cells in the presence of IL-2.
 59. A method forobtaining a population of antigen-specific regulatory T cells withcytotoxic properties, the method comprising the steps of: providing animmunogenic peptide according to claim 37, administering said peptide toa subject, and obtaining said population of antigen-specific regulatoryT cells from said subject.
 60. The population of T regulatory cellsobtainable by the method of claim 58 for use in the treatment andprevention of an allergic condition or an auto-immune disorder.
 61. Thepopulation of T regulatory cells obtainable by the method of claim 59for use in the treatment and prevention of an allergic condition or anauto-immune disorder.